Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts

BioMed Research International

BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts

Guest Editors: Ameur Cherif, George Tsiamis, Stéphane Compant, and Sara Borin BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts BioMed Research International BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts

This is a special issue published in “BioMed Research International.” All articles are open access articles distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Contents

BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts,AmeurCherif, George Tsiamis, Stephane´ Compant, and Sara Borin Volume 2015, Article ID 289457, 2 pages

The Date Palm Tree Rhizosphere Is a Niche for Plant Growth Promoting Bacteria in the Oasis Ecosystem, Raoudha Ferjani, Ramona Marasco, Eleonora Rolli, Hanene Cherif, Ameur Cherif, Maher Gtari, Abdellatif Boudabous, Daniele Daﬀonchio, and Hadda-Imene Ouzari Volume 2015, Article ID 153851, 10 pages

Pentachlorophenol Degradation by Janibacter sp., a New Actinobacterium Isolated from Saline Sediment of Arid Land, Amel Khessairi, Imene Fhoula, Atef Jaouani, Yousra Turki, Ameur Cherif, Abdellatif Boudabous, Abdennaceur Hassen, and Hadda Ouzari Volume 2014, Article ID 296472, 9 pages

Biotechnological Applications Derived from Microorganisms of the Atacama Desert, Armando Azua-Bustos and Carlos Gonzalez-Silva´ Volume 2014, Article ID 909312, 7 pages

Diversity and Enzymatic Proﬁling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia, Atef Jaouani, Mohamed Neifar, Valeria Prigione, Amani Ayari, Imed Sbissi, Sonia Ben Amor, Seifeddine Ben Tekaya, Giovanna Cristina Varese, Ameur Cherif, and Maher Gtari Volume 2014, Article ID 439197, 11 pages

Geodermatophilus poikilotrophi sp. nov.: A Multitolerant Actinomycete Isolated from Dolomitic Marble, Maria del Carmen Montero-Calasanz, Benjamin Hofner, Markus Goker,¨ Manfred Rohde, Cathrin Sproer,¨ Karima Hezbri, Maher Gtari, Peter Schumann, and Hans-Peter Klenk Volume 2014, Article ID 914767, 11 pages

Safe-Site Eﬀects on Rhizosphere Bacterial Communities in a High-Altitude Alpine Environment, Sonia Ciccazzo, Alfonso Esposito, Eleonora Rolli, Stefan Zerbe, Daniele Daﬀonchio, and Lorenzo Brusetti Volume 2014, Article ID 480170, 9 pages

Contrasted Reactivity to Oxygen Tensions in Frankia sp. Strain CcI3 throughout Nitrogen Fixation and Assimilation, Faten Ghodhbane-Gtari, Karima Hezbri, Amir Ktari, Imed Sbissi, Nicholas Beauchemin, Maher Gtari, and Louis S. Tisa Volume 2014, Article ID 568549, 8 pages

Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils, Okba Selama, Gregory C. A. Amos, Zahia Djenane, Chiara Borsetto, Rabah Forar Laidi, David Porter, Farida Nateche, Elizabeth M. H. Wellington, and Hocine Hacene` Volume 2014, Article ID 317524, 11 pages

Absence of Cospeciation between the Uncultured Frankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species, Imen Nouioui, Faten Ghodhbane-Gtari, Maria P. Fernandez, Abdellatif Boudabous, Philippe Normand, and Maher Gtari Volume 2014, Article ID 924235, 9 pages Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 289457, 2 pages http://dx.doi.org/10.1155/2015/289457

Editorial BIODESERT: Exploring and Exploiting the Microbial Resource of Hot and Cold Deserts

Ameur Cherif,1 George Tsiamis,2 Stéphane Compant,3 and Sara Borin4

1 University of Manouba, Biotechnology and Bio-Geo Resources Valorization (LR11-ES31), Higher Institute for Biotechnology, BiotechPole Sidi Thabet, 2020 Ariana, Tunisia 2Department of Environmental and Natural Resources Management, University of Patras, 2 Seferi Street, 30100 Agrinio, Greece 3Bioresources Unit, Health & Environment Department, AIT Austrian Institute of Technology GmbH, 3430 Tulln, Austria 4DepartmentofFood,EnvironmentalandNutritionalSciences(DeFENS),UniversityofMilan,ViaCeloria2,20133Milan,Italy

Correspondence should be addressed to Ameur Cherif; [email protected]

Received 15 February 2015; Accepted 15 February 2015

Copyright © 2015 Ameur Cherif et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Deserts are generally regarded as lifeless and inhospitable communities shape and dynamic and the functional net- ecosystems despite the general awareness of extremophilic working including mechanism of adaptation and plant- microorganisms. An amazing microbial diversity and a huge microbes interaction under extreme or changing conditions, biotechnological potential were unraveled during the last and (c) potential and case applications of desert microbes decade using molecular approaches. Hot and cold deserts and/or mixed cultures, such as in soil bioreclamation, wereshowntohostpeculiarmicrobialassemblagesableto reverse-desertification, agriculture, and biomining. cope with hostile environment and/or to rapidly adapt to This special issue contains one review and eight research changing conditions. This adaptation is inferred to partic- articles that address the three main aspects indicated above. ular community structure behavior and specific metabolic The paper of A. Jaouani et al., entitled “Diversity and Enzy- capacities allowing cells to overcome water stress, fluctuating matic Profiling of Halotolerant Micromycetes from Sebkha El temperature, and high salinity. Therefore, such microbes Melah, a Saharan Salt Flat in Southern Tunisia,” reported the could constitute a source of novel metabolites, biomolecules, isolation of 21 alkali-halotolerant Ascomycetes assigned to the and enzymes potentially useful for environmental biotech- 6 genera Cladosporium, Alternaria, Aspergillus, Penicillium, nologies.Withtheglobalclimatechange,thearidificationand Ulocladium, and Engyodontium, basing on morphological creeping desertification that constitute a worldwide serious and molecular markers. Beside their salt and pH tolerance, threat directly affecting agriculture and crop production, and these saline-system fungi were shown to resist to oxidative the growing food demands, desert microorganisms could stress and low temperature and to produce extremozymes, hold the key for green biotechnology and future applications namely,cellulase,amylase,protease,lipase,andlaccase,active into soil bioreclamation and plant growth promotion for in high salt concentrations, which highlight their biotechno- vulnerable regions across the world. logical potential. The paper authored by M. del C. Montero- This special issue was focused on the desert microbial Calasanz et al., “Geodermatophilus poikilotrophi sp. nov.: A resource management (MRM) and how to explore and exploit Multitolerant Actinomycete Isolated from Dolomitic Marble,” these resources from hot and cold deserts as well as from described a new species within the genus Geodermatophilus. arid areas. Aspects of this MRM concept are included in Strain G18T, isolated from site near the Namib Desert, this special issue and they are highlighting (a) the microbial is characterized by its resistance to heavy metals, metal- diversity and community structure behavior in desert envi- loids, hydrogen peroxide, desiccation and ionizing, and UV- ronments and the identification of novel extremophiles, (b) radiations. Even though 16S rRNA sequence of strain G18T the influence of the biotic and abiotic factors on microbial showed 99% similarity with other Geodermatophilus species, 2 BioMed Research International its taxonomic position and species definition was inferred the prerequisite of providing rhizosphere services and specific basing on polyphasic approach and its multitolerance towards functionalities. environmental stresses, justifying the original given epithet Biotechnological potential and applications of desert “poikilotrophi.” microbes have been reported in three papers. In one, A. Four papers were dedicated to the ecological drivers Khessairi et al. described a novel efficient pentachlorophenol- that shape microbial communities and functionalities. A (PCP-) degrading halotolerant actinobacterium, Janibacter nice example of “cold desert” is presented by S. Ciccazzo et sp.FAS23.ThestrainwasisolatedfromSebkhaElNaoual,a al., “Safe-site Effects on Rhizosphere Bacterial Communities saline ecosystem in southern Tunisia. Using HPLC analysis, in a High-Altitude Alpine Environment.” In this work, the FAS23 was shown to be able to degrade high concentration authors investigated rhizobacterial communities associated of PCP (up to 300 mg/l) and to tolerate salt fluctuation. PCP with floristic consortia in different safe-sites located in degradation was further enhanced in the presence of glucose deglaciated terrain. Using DGGE and ARISA, they demon- and nonionic surfactant tween 80. The strain is considered as strated a clear correlation between soil maturation and a candidate for PCP bioremediation in polluted soils in arid bacterial diversity and a plant-specific effect leading to the areas. In another paper authored by O. Selama et al., the iso- selection of specific rhizobacterial communities by the pio- lation of haloalkalitolerant and haloalkaliphilic bacteria from neer plants. Another model ecosystem was investigated by R. Algerian Sahara Desert soil was reported. Thirteen selected Ferjanietal.,inthepaper“TheDatePalmTreeRhizosphere isolates, mainly filamentous Actinobacteria,werescreened Is a Niche for Plant Growth Promoting Bacteria in the Oasis phenotypically for antibacterial, antifungal, and enzymatic Ecosystem.” The work focused on the characterization of activities and by PCR for putative antitumor compounds the bacterial communities in the soil fractions associated genes. The isolates were assigned to the genera Streptomyces, with the root system of date palms cultivated in seven Nocardiopsis, Pseudonocardia, Actinopolyspora, and Nocar- oases in Tunisia using culture-independent and dependent dia, with this latter constituting possibly a new branch in approaches. It was shown that the date palm rhizosphere the Actinomycetales order. Beside secreted extremozymes and bacterial communities were rather complex and correlate bioactives, several isolates showed antitumorigenic potential. with geoclimatic and macroecological factors along a north- Another paper presented in this special issue is a review arti- south aridity transect. However, with the wide diversity of cle nicely written by A. Azua-Bustos and C. Gonzalez-Silva,´ cultivable strains detected, interesting common features of who focused on the Atacama Desert microbes and their cur- plant growth promoting (PGP) activity and abiotic stress rent biotechnological applications. A large-scale application resistance were detected. The authors concluded that palm in Chile is the copper bioleaching or biomining mediated by root system and rhizosphere soil represent a reservoir of PGP indigenous halotolerant and acidophilic chemolithotrophic bacteria involved in the regulation of plant homeostasis. The bacteria like Acidithiobacillus ferrooxidans and Acidithiobacil- third example of plant-microbe interaction is the paper by lus thiooxidans. Other potential applications in arsenic biore- I. Nouioui et al., in which they demonstrated, as written in mediation and in biomedicine, including the discovery of thetitle,the“AbsenceofCospeciationbetweentheUncul- new antibiotics, antioxidant, antifungal, and immunosup- tured Frankia Microsymbionts and the Disjunct Actinorhizal pressive compounds, were cited. The authors reported also Coriaria Species.” The investigation was achieved on five application from eukaryotic microorganism as in the case Coriaria host species sampled from sites covering the full of the halophilic biflagellate unicellular green alga Dunaliella geographical range of the genus (Morocco, France, New that produce beta-carotene. Zealand, Pakistan, Japan, and Mexico). The reported find- Definitely, desert environments represent a tremendous ings argue that Frankia, the nitrogen-fixing actinobacteria reservoir where more efforts, relying not only on metage- microsymbionts, have not evolved jointly with their host nomics but also on culturomics, should be dedicated to plants and had probably dispersed globally as a proto- unravel the hidden potential. The second decade for desert Frankia, a free living nonsymbiotic ancestor. The authors biotechnology has just begun. hypothesized also that cospeciation may have occurred but subsequently lost after bacteria mixing and fitness selection in Acknowledgments thepresenceofindigenoussymbionts.Frankia sp. was further investigated in terms of nitrogen fixation under different We thank the authors of the submitted papers for their oxygen tensions. This work authored by F. Ghodhbane-Gtari contribution. The preparation of this special issue would et al. was conducted on the actinorhizal plant Casuarina and nothavebeenpossiblewithoutthegeneroussupportand its compatible Frankia sp. strain CcI3. By studying the growth dedication of experts who evaluated the papers submitted. ofthestrain,vesicleproduction,andseveralgenesexpression, the authors confirmed the correlation between the biomass Ameur Cherif andthevesicleproductionwithelevatedoxygentension.It George Tsiamis was also shown that oxygen levels influenced nitrogenase Stephane´ Compant induction and that Frankia protects nitrogenase by the use of Sara Borin multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection. Clearly, the microbial assemblages selected by the plant roots in desert and arid soils areshapedbytheecologicalbioticandabioticdriversbutwith Hindawi Publishing Corporation BioMed Research International Volume 2015, Article ID 153851, 10 pages http://dx.doi.org/10.1155/2015/153851

Research Article The Date Palm Tree Rhizosphere Is a Niche for Plant Growth Promoting Bacteria in the Oasis Ecosystem

Raoudha Ferjani,1 Ramona Marasco,2 Eleonora Rolli,3 Hanene Cherif,1 Ameur Cherif,4 Maher Gtari,1 Abdellatif Boudabous,1 Daniele Daffonchio,2,3 and Hadda-Imene Ouzari1

1 LR03ES03 Laboratoire Microorganismes et Biomolecules´ Actives, Faculte´ des Sciences de Tunis, UniversitedeTunisElManar,´ Campus Universitaire, 2092 Tunis, Tunisia 2 Biological and Environmental Sciences and Engineering Division, King Abdullah University of Science and Technology, Thuwal 23955-6900, Saudi Arabia 3 Department of Food, Environment, and Nutritional Sciences, University of Milan, Via Celoria 2, 20133 Milan, Italy 4 UniversitedeLaManouba,InstitutSup´ erieur´ de Biotechnologie de Sidi Thabet, LR11ES31 LR Biotechnologie & Valorisation des Bio-Geo´ Ressources, BiotechPole Sidi Thabet, 2020 Ariana, Tunisia

Correspondence should be addressed to Hadda-Imene Ouzari; [email protected]

Received 16 May 2014; Accepted 6 October 2014

Academic Editor: Sara Borin

Copyright © 2015 Raoudha Ferjani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In arid ecosystems environmental factors such as geoclimatic conditions and agricultural practices are of major importance in shaping the diversity and functionality of plant-associated bacterial communities. Assessing the influence of such factors is a key to understand (i) the driving forces determining the shape of root-associated bacterial communities and (ii) the plant growth promoting (PGP) services they provide. Desert oasis environment was chosen as model ecosystem where agriculture is possible by the microclimate determined by the date palm cultivation. The bacterial communities in the soil fractions associated with the root system of date palms cultivated in seven oases in Tunisia were assessed by culture-independent and dependent approaches. According to 16S rRNA gene PCR-DGGE fingerprinting, the shapes of the date palm rhizosphere bacterial communities correlate with geoclimatic features along a north-south aridity transect. Despite the fact that the date palm root bacterial community structure was strongly influenced by macroecological factors, the potentialhizosphere r services reflected in the PGP traits of isolates screened in vitro were conserved among the different oases. Such services were exerted by the 83% of the screened isolates. The comparable numbers and types of PGP traits indicate their importance in maintaining the plant functional homeostasis despite the different environmental selection pressures.

1. Introduction overexploitation and strong anthropogenic pressures, these ecosystems are becoming increasingly fragile. Furthermore, The southern regions of Tunisia are very arid and the date despite the oasis potential to tolerate several abiotic stresses palm (Phoenix dactylifera L.) is a key plant determining in typical of arid environment, the ongoing climate change the oasis agroecosystem a microclimate that favours agri- is enhancing the environmental pressure on the date palm culture [1]. The palms protection provides many ecosystem affecting growth and development, especially in the Middle services, including ameliorating oasis temperature, changing East [5]. floodwater dynamics and facilitating wildlife, and making Besides the well-known plant growth promoting prop- agriculture possible under harsh environmental conditions erties typical of rhizospheres in temperate soils in nonarid [2].Intheworld,oasescoverabout800000haandsupport ecosystems, rhizosphere bacteria in arid soils contribute in the living of 10 million people. In Tunisia more than four mil- counteracting drought and salinity stresses, by providing lionsofdatepalmtreesarespreadonto32000haofoasisin servicessuchas,amongothers,physicalprotectionofthe the southern part of the country [3, 4]. As a result of the oases root from mechanical stress against the dry soil particles, 2 BioMed Research International induction of plant physiological responses against water according to the manufacturer’s procedure. PCR amplifica- losses [6], or productions of metabolites contributing to the tion was performed in a final volume of 50 𝜇Lusingprimers maintenance of the plant hormone and nutrient homeostasis, 907R and 357F, adding a GC-clamp to the forward primer [7]. In particular PGP (plant growth promoting) bacteria, [14, 15]. The reaction mixture was prepared with 1X PCR naturallyassociatedwithplants,havebeenshowntobe buffer, 2.5 mM MgCl2, 0.12 mM deoxynucleoside triphos- essential partners for improving plant tolerance to stressful phate, 0.3 mM of each primer and 1U Taq DNA polymerase conditions [8]. The exploration of plants naturally adapted and10ngofpooledDNAobtainedfromthethreeplantrepli- to extreme condition may allow a reservoir of biodiversity cates were added as template. PCR products were resolved on exploitable to understand the ecological service enclosed in 7% (w/v) polyacrylamide gel in 1X TAE pH 7.4with a 40–60% ∘ these ecosystems [8, 9]. In this context, ecological niche pre- denaturing gradient. Gels were run at 90 V for 17 h at 60 C sented in the oasis ecosystem could provide a new model to in DCode apparatus (Bio-Rad, Italy). After electrophoresis, study and dissect the key factors driving the stability of this gels were stained with ethidium bromide solution for 30 min, ecosystem [10]. Little information is available about the washed with sterile distilled water, and photographed on a microbiological functionality of both oasis and date palm. UV transillumination table. The DGGE band profiles were For instance the potential PGP services provided by the root- converted into numerical values using Image J (version 1.46) associated bacteria appear to be invariant with respect to and XLSTAT software. geoclimatic factors despite provided by different bacterial communities, according to observations across a north to 2.3. Real Time PCR. Quantitative real time PCR (q-PCR) south aridity transect that included Tunisia [11]. was performed on a Chromo4 real time PCR machine (Bio- Since plants contribute to shape soil microbial diversity Rad) to measure the presence and concentration of bacterial [12, 13], the aim of this work was to assess bacterial com- 16S rRNA gene associated with the rhizosphere fractions. munities associated with the date palm rhizosphere soil, the The reactions were performed with IQ SYBR Green Super- root surrounding soil and the bulk soil fractions in seven mix (Bio-Rad), using primers targeting the 16S rRNA gene Tunisian oases, in order to evaluate if along a north-south (Bac357-F and Bac907-R) [16]. PCR SYBR green reactions transect (i) the assemblage of bacterial communities in the were prepared by using the “Brilliant SYBR Green QPCR palm root soil fractions was driven by the geoclimatic factors Master Mix” kit (Stratagene) in 96-well plates. The reaction and(ii)theecologicalserviceswerepreservedinthesoil mix (25 mL) contained 1X Brilliant SYBR Green (2.5 mM fractions of the root system. The structure of the bacterial MgCl2), 0.12 mM of each primers, and approximately 100 ng communitiesassociatedwiththesoilfractionsofdatepalmin of extracted DNsA. The DNA obtained from the three plants the seven oases was dissected by 16S rRNA gene-based PCR- sampled in the same station was pooled and used as template DGGE (denaturing gradient gel electrophoresis) analysis. The to carry out the real time assay in triplicate. At the end of results were analysed in function of geoclimatic factor and each real time PCR, a melting curve analysis was performed oasis origin, and compared with the diversity of the cultivable for verifying the specificity of PCR products. To construct bacteria and their PGP potential. standard curves, the 16S rRNA gene of Asaia sp. was amplified by PCR and cloned using the pGEM T-easy Vector Cloning 2. Materials and Methods Kit (Promega). q-PCR data relative to the 16S rRNA gene concentration were log-transformed. 2.1. Site Description and Sampling. The sampling was carried out from seven oases in different geographic locations in 2.4. Isolation of Cultivable Bacteria. One gram of rhizosphere Tunisia, along a latitude/longitude gradient, respectively from ∘ ∘ ∘ ∘ soil (R) from each sample was suspended in 9 mL of sterile 32 to 34 Nandfrom7 to 9 E(Figure 1(a) and Supplemen- physiological solution (9 g/L NaCl) and shaken for 15 min tary Table 1 in the Supplementary Material available online at at 200 rpm at room temperature. Suspensions were diluted http://dx.doi.org/10.1155/2015/153851). A traditional crop man- in tenfold series and plated in triplicate onto TSA (Tryptic agement was used in all the oases, including groundwater- Soy Agar), YEM (Yest Extract Mannitol), and KB (King’B ∘ based flooding irrigation and fertilization with organic fer- agar) culture media. After three days at 30 Ccolonieswere tilizers. In each oasis, the roots of three date palm trees of randomly selected and spread on the original medium for similar age, lying in the distance of less than 15 meters and three times to avoid contamination risks. Pure strains were ∘ growing in the same soil were separately collected at 20– frozen in 25% glycerol at −80 C. A total of 440 isolates were 30 cm depth in order to obtain the adhering rhizosphere soil collected. The isolates were named based on the station and (R) tightly attached to roots. After removing the roots, the the medium from which they were isolated. rootsurroundingsoil(S)wascollected.Bulksoilsamples(B) not influenced by date palm root system were also sampled 2.5. DNA Extraction, Dereplication, and Identification of Iso- as control. All soil samples were collected under sterile lates. Genomic DNA was recovered from the isolates using a conditions using sterile tools. Recovered samples were stored 𝜇 ∘ ∘ boiling lysis. Bacterial cells were suspended in 50 Lofsterile at −20 Cformolecularanalysisorat4Cforisolation. TE (10 mM Tris/HCl, pH 8, 1 mM EDTA) and incubated ∘ at 100 C for 8 min. After centrifugation (13000 g, 10 min), 2.2. Total DNA Extraction, PCR-DGGE, and Profile Analysis. the supernatant containing the released DNA was stored at ∘ Total DNA from soil samples was extracted by commercial −20 C and used as template for PCR. Amplification of 16S– kit FASTDNA SPIN KIT for soil (Qbiogene, Carlsbad, USA) 23S internal transcribed spacer region (ITS) was performed BioMed Research International 3

20 R S B R R R B R S S 0 R S R S % of total variation) total % of 6 . B B S

22 B ( 2 S −20 PCO −40 −20 0 20 40 Sampling station PCO1 (62.2% of total variation) Ksar Ghilane (BD-1) Tozeur (BD-16) Douz (BD-5) Tamerza (BD-B) El Faouar (BD-8) Ain el Karma (BD-C) Rjim Maatoug (BD-9) (a) (b) 10 7.5

5 7.0 a T (∘ ) min C ab a a 2 Rainfall max (mm) a Long. E

% of total variation) total % of 0 6.5 1 . ∘ Lat. N 7 T ( C)

dbRDA max

S rRNA copies) S rRNA a 6.0 b Rainfall min (mm) 16 −5 ( 10

% of fitted, 5.5

Alt (m) Log 2 . 16 ( −10 5.0 −10−50 5 101520 BD-1 BD-5 BD-8 BD-9 BD-16 BD-B BD-C dbRDA1 (68.1% of fitted, 29.9% of total variation) (c) (d) Figure 1: Station location and analysis of bacterial community structure associated with soil fraction of date root system. (a) The location of the studied oases is indicated on the map of Tunisia. (b) A principal coordinate analysis (PCO), deduced from the 16S rRNA gene-based PCR- DGGE profiles, resumes the diversity of the bacterial communities associated with the date palm root soil fractions. 84.8% of total variation is explained in the presented PCO. The soil fractions analysed are R: rhizosphere; S: root surrounding soil; and B: bulk soil. (c) Dist LM analysis to evaluate which are the main geoclimatic variables influencing the structure of the bacterial communities associated with date palm root soil fractions. Lat. N: latitude north; Long. E: longitude east; Alt.: altitude; 𝑇min: minimum temperature; 𝑇max: maximum temperature; Rainfall min: minimum rainfall; Rainfall max: maximum rainfall. (d) Box-plot graph represents the quantification of 16S rRNA gene by qPCR. The number of copies is expressed in Log10. Statistical analysis (pairwise test) of bacterial assemblage across locations was indicated by the letter.

󸀠 using the universal primers S-DBact-0008-a-S-20 (5 -CTA Shrimp Alkaline Phosphatase (Exo-Sap, Fermentas, Life 󸀠 CGG CTA CCT TGT TAC GA-3 ) and S-D-Bact-1495-a-S-20 Sciences) following the manufacturer’s standard protocol. 󸀠 󸀠 (5 -AGA GTT TGA TCC TGG CTC AG-3 ) according to the Sequencing of the purified amplicons was performed using procedure described previously by Fhoula et al. [17]. Two 𝜇L a Big Dye Terminator cycle sequencing kit V3.1 (Applied of the PCR products were checked by electrophoresis in 1.5% Biosystems) and an Applied Biosystems 3130XL Capillary agarose gel and stained with ethidium bromide. Gel images DNA Sequencer machine. The obtained sequences, with an were captured using Gel Doc 2000 system (Bio-Rad, Tunis, average length of 750 bp, were compared with those available Tunisia), and bacteria redundancy was reduced by evaluating at the National Centre for Biotechnology Information (NCBI) the different ITS profiles. One strain per each ITS haplotype database (http://www.ncbi.nlm.nih.gov)usingthebasiclocal was used in the phylogenetic analysis and for further experi- alignment search tool (BLAST) algorithm [18]. The 16S rDNA ments. A total of 98 strains were characterized by 16S rRNA sequences were submitted to the NCBI nucleotide database 󸀠 gene sequencing using the primers S-D-Bact-1494-a-20 (5 - under the accession number KJ956590 to KJ956687. 󸀠 GTC GTA ACA AGG TAG CCG TA-3 ) and L-D-Bact-0035- Phylogenetic analysis of the 16S rRNA gene sequences 󸀠 󸀠 a-15 (5 -CAA GGC ATC CAC CGT-3 ). PCR amplification was conducted with molecular evolutionary genetics analysis was carried out as described by Fhoula et al. [17]. The 16S (MEGA) software, version 619 [ ]. Trees were constructed by rRNA PCR amplicons were purified with Exonuclease-I and using neighbor-joining method [20]. 4 BioMed Research International

2.6. Characterization of Plant Growth Promoting Activity and bacterial communities associated with the date palm root Abiotic Stress Resistance. The 98 bacterial strains identified system from each of the seven studied oasis was investigated were screened for production of indole acetic acid (IAA), through the analysis of the diversity of the 16S rRNA gene in siderophores and ammonia, mineral phosphate solubiliza- the rhizosphere (R) and root surrounding soil (S) fractions. tion,proteaseandcelluloseactivity,andtolerancetosev- Bulk soil (B) was also included as a comparative fraction not eral abiotic stresses. Quantitative production of IAA was directly influenced by the plant root. Separation among determined as described by Ouzari et al. [21]. Briefly, after palm bacterial communities located in north (BD-16, BD- incubation in minimal medium supplemented with glucose B, and BD-C) and south (BD-1, BD-5, BD-8, and BD- (100 g/L) and L-tryptophane (10 𝜇g/mL), using Salkowski’s 9) oases was supported by a principal coordinate analysis reagent,thecolourabsorbancewasread,after20–25min,at (PCO) (Figure 1(b)), suggesting that geoclimatic conditions 535 nm. Concentration of IAA produced was measured by influence the bacterial community structure. Statistical anal- comparison with a standard graph of IAA. Ability of bacteria ysis confirmed the grouping observed in the PCO analysis to solubilize inorganic phosphate was evaluated as described with a significant difference between north and south oases by Nautiyal [22], by the observation of clear halo around the (PERMANOVA, df = 1.55; 𝐹 = 8.06; 𝑃 = 0.0017)butnota bacterial colony grown in Pikovskaya medium. To demon- significant separation mediated by the aquifer used to irrigate strate the production of siderophore, the tested strains were the oases (PERMANOVA, df = 1.55; 𝐹 = 1.45; 𝑃 = 0.21). spottedonnutrientagarplates.Afterincubationfor48–72h Within the two macroregions, the north and south ∘ at 30 C, the grown strains were overlaid with CAS medium groups of oases, we observed a significant difference among supplemented with agarose (0.9% w/v). Positive test was oases (Supplementary Table 2) indicating the presence of noted when colour modification around colonies from blue to oasis-specific bacterial community supporting a concept of orange was observed [23]. Ammonia production was assayed “ecological island.” Pairwise analysis showed that such dif- by inoculation of bacterial strains in 10 mL of peptone water ferences observed among the oases predominantly occurred and using Nessler’s reagent (0.5 mL). Ammonia producing in the north regions (Supplementary Table 3), possibly strainswereidentifiedwhenbrowntoyellowcolourwas because the south region (closest to desert) presents harsher developed [24]. Protease (casein degradation) and cellulase conditions that select a more restricted type of bacteria. These activities were determined by spot inoculation of the strains ecological islands represent specific cluster of biological on Skimmed milk and CMC agar media, respectively. A clear diversity that may contribute to the overall regional bacterial halo around the colonies indicates the ability of the strains to community functionality and furthermore increase the level produce the degrading enzymes [25]. of resilience to environmental change of the entire system Tolerance to osmotic stress was evaluated by adding to [30]. tryptic soy broth (TSB) medium 30% of polyethylene glycol Along the transect, the soil fraction communities were (PEG 8000). Resistance to salt was assessed by adding 5, 10, significantly different (PERMANOVA, df = 2.55; 𝐹 = 2.70; 15,20,25,and30%NaCltotheculturemediaandincubating 𝑃 = 0.03). In particular the rhizosphere community, that ∘ the plates at 30 Cfor5days.Theabilitytogrowthat45,50, resides in the first millimeter of soil adhering to the root, ∘ and 55 C was checked in TSA by incubation at the indicated appeared completely different from the root surrounding soil temperatures for 5 days. Tolerance to acid (3 and 4) and basic (PERMANOVA, 𝑡 = 2.04; 𝑃 = 0.017,p-pht)andbulksoil (10and12)pHwasassessedbyadjustingthemediumwith (PERMANOVA, 𝑡 = 2.05; 𝑃 = 0.019, p-pht), suggesting the concentrated HCl (12 N) and NaOH (3 M), respectively. influence of palm root exudates in shaping the bacterial community. Generally, the rhizosphere is the transition zone 2.7. Statistical Analysis. Significant differences in soil bacte- between the root surface and soil where the released exu- rial community structure were investigated by permutational dates and the rhizodeposition favour microbial proliferation, analysis of variance (PERMANOVA, [26]). Distance-based inducing changes in the structure and in the chemical-physi- multivariate analysis for a linear model (DistLM, [27]) was cal properties of the soil [31]. Indeed, the analysis of bacterial used to determine which significant environmental variables abundanceintherhizosphereshowedanumbersof16SrRNA explain the observed similarity among the samples. The copies ranging from 5.88 ± 0.78 to 6.63 ± 0.15 (Figure 1(d)). Akaike information criterion (AIC) was used to select the Despite similar values observed in the rhizosphere commu- predictor variables [28]. The contribution of each environ- nity, a statistical difference among the stations was identified mental variable was assessed: firstly the “marginal test” is used (PERMANOVA, df = 6.20; 𝐹 = 2.93; 𝑃 = 0.041), mainly to assess the statistical significance and percentage contribu- influenced by environmental factor directly linked to location of each variable by itself and secondly the “sequential tion, such as altitude and temperature maximum (DistLM, test” was employed to explain the biotic similarity consid- 𝑃 = 0.03). eringallthevariablecontributions.Allthestatisticaltests Despitetherhizosphereeffectobservedalongthetran- were performed by PRIMER v. 6.1 [29], PERMANOVA+for sect, in each oasis considered separately from the others, rhi- PRIMER routines [30]. zobacterial community appeared directly connected to that present in the root surrounding soil and the bulk soil, since 3. Results and Discussion no statistically significant differences in the bacterial diversity were observed among the different soil fractions within each 3.1. Environment Parameters Directly Influence Bacterial Com- station (R, S e B: PERMANOVA, df = 12.55; 𝐹 = 1.62; 𝑃= munities Associated with Palm Rhizosphere. The diversity of 0.057). The rhizosphere effect is particularly noticeable in BioMed Research International 5

Table 1: Environmental factors associated to the structure of the date palm soil bacterial community. Relationships between bacterial assemblages and climate features using nonparametric multivariate multiple regression analysis (DISTLM). (a) Marginal test considers each single geographical variables and their contribution to explain the total variability. (b) Sequential test explaining the total variation with the contribution of all the variables accounted together. Lat. N: latitude north; Long. E: longitude east; Alt.: altitude; 𝑇min: minimum temperature; 𝑇max: maximum temperature; Rainfall min: minimum rainfall; Rainfall max: maximum rainfall; 𝐹:statistic𝐹; 𝑃:probability(inboldthe variables statistically significant; 𝑃 < 0.05); Prop.: proportion of total variation explained; Cumul.: cumulative variation explained by the variables listed; Res df: residual degrees of freedom.

(a) Marginal test

Variable SS (trace) 𝐹𝑃 Lat. N 1855.5 6.4211 0.0028 Long. E 1444.3 4.8698 0.0097 Alt (m) 980.15 3.2116 0.0376 ∘ 𝑇min ( C) 1170.8 3.8811 0.0193 ∘ 𝑇max ( C) 363.14 1.147 0.3034 Rainfall min (mm) 1187.3 3.9401 0.0198 Rainfall max (mm) 588.06 1.8821 0.136 (b) Sequential test Variable AIC SS 𝐹𝑃Cumul. Res. df (+) Lat. N 319.28 1855.5 6.4211 0.0022 0.10627 54 (+) Long. E 320.07 331.89 1.1517 0.2998 0.12528 53 (+) Alt (m) 317.33 1240.6 4.5974 0.0122 0.19633 52 ∘ (+) 𝑇min ( C) 316.7 644.65 2.4558 0.084 0.23325 51 ∘ (+) 𝑇max ( C) 309.31 2066 9.1244 0.0006 0.35158 50 (+) Rainfall min (mm) 303.19 1528.3 7.6468 0.001 0.43911 49 (+) Rainfall max (mm) 303.19 < 0.01 0 1 0.43911 49 nutrient-poor soils and under severe abiotic stresses, as pre- a concurrence of environmental factors, including a hot and viously observed for herbaceous and arboreal plants grown dry climate, may influence the differences among the bacte- in arid lands [7, 8, 32, 33]. In the oasis model the selection rialcommunitiesofthesoilfractions(R,S,andB)associated mediated by “oasis ecosystem” appeared stronger than the with the root system of date palm cultivated in the oases in one exerted by the plant root system (Supplementary Table the north and south macroregions examined (Figure 1(c)). 2). Naturally, most of the desert microbial communities seem to be structured solely by abiotic processes [34, 35]. However, 3.2. Cultivable Bacterial Communities Associated with Date desert farming may strongly affect the sand/soil microbial Palm Soil Fractions. The isolation of native bacterial species diversity reshaping the structure of the resident microbial associated with date palm root soil was performed using communities [8, 9, 36, 37]. During long-term desert farming nonspecific media, in order to select a wide range of genera land management, such as that occurring in the studied oases, of possible plant growth promoters [39–41]. thestructureofrhizospherebacterialcommunityisstrongly A total of 440 isolates were retrieved from the seven influenced by the plant and the desert farming practices that analyzed stations. To manage such a large set of isolates, total determine drastic shifts in the composition of the original DNA was extracted from each isolate and 16S–23S rRNA desert soil communities [7, 8]. gene internal transcribed spacers (ITS) were amplified. ITS Dist LM multivariate analysis was performed in order to characterization represents a useful molecular tool for the correlate the differences in the structure of bacterial commu- discrimination of bacterial isolates up to the subspecies level nities in the different oases with environmental parameters. [42–45]. Within the whole bacterial collection, ITS-PCR The selection of soil microorganisms by the rhizosphere is a fingerprinting revealed 30 distinct haplotypes (H1-H30). complex process controlled by several factors, often not easily Haplotypes H4 and H20 were the most frequent and were correlated to the environmental settings [38]. Nevertheless, represented by 46 and 26 isolates, respectively. Representa- Dist LM analysis showed that geoclimatic parameters con- tive isolates (from one to four strains for each haplotype, tributedtodrivetheassemblageofthebacterialcommunities. summing up a total of 98 isolates) were subjected to species In particular, marginal test showed latitude, longitude, alti- identification using partial 16S rRNA gene sequencing (Sup- tude, minimum temperature, and minimum rainfall as signif- plementary Figure 1). icant variables singularly involved in the selection of bacterial Awidediversitywasdetectedintodatepalmrhizo- assemblages (Table 1(a)). Sequential test confirmed latitude, sphere bacterial community along the studied aridity tran- altitude, and temperature as variables involved in the bac- sect in Tunisia. Significant differences were observed in the terial community shaping (Table 1(b)). We can assume that structure of the bacterial communities in the rhizosphere of 6 BioMed Research International theanalyzedoases,inparticularforthedifferentialdistri- 100 bution pattern of the major bacterial genera (Figure 2(a)). 90 According to the cluster analysis at the genus level performed 80 on the entire strain collection, the composition of the cultivable rhizobacterial communities associated with date palm 70 inthesevenoasessharedaboutthe65%similarity. 60 The phylogenetic identification of cultivable bacte- 50 ria highlighted a predominance of gram-negative bacte- 40 ria (66%), belonging to the Gammaproteobacteria (57%), (%) Isolates Alphaproteobacteria (7%), and Betaproteobacteria (1%) sub- 30 classes. The remaining isolates were affiliated to the Fir- 20 micutes (7%), Actinobacteria (26%), and Bacteroidetes (2%) 10 classes. Members of these taxa are frequently associated with 0 different plant species and types, confirming that soil is the BD-1 BD-5 BD-8 BD-9 BD-16 BD-B BD-C main reservoir of plant-associated bacteria [46]. The strains were allocated into 20 different genera of variable occurrence Streptomyces Bacillus Providencia (Figure 2(a)), showing a high genetic diversity in the date Arthrobacter Agrobacterium Yersinia palmrhizospherepresumablyinfluencedbythecombined Labdella Thalassospira Rahnella Mycobacterium Rhizobium Salinicola effects of root exudates and agricultural management prac- Cellulomonas Flavobacterium Enterobacter tices, particularly important under the arid pedoclimatic Microbacterium Pantoea Pseudomonas conditions [47]. The rhizobacterial communities were domi- Staphylococcus Serratia nated by Pseudomonas, as previously described in herbaceous (a) plants, arboreal and plant adapted to arid climates [11, 48–50]. Together with Pseudomonas, Pantoea and Microbacte- 100 rium genera were observed in all stations followed by Bacillus 80 and Arthrobacter, which were reported in six out of seven stations. As well, Enterobacter, Salinicola, Rhizobium,andStaph- 60 ylococcus were recorded among 5 stations, suggesting the 40

adaptation of these genera to the oasis environment. Except (%) Isolates Labedella,thegenerafoundinassociationwithdatepalm 20 rhizospherehavebeenpreviouslyrecognizedasbeingcapable of colonizing plant root systems in arid environment [11, 38, 0 48, 50–52]. BD-1 BD-5 BD-8 BD-9 BD-16 BD-B BD-C P Solubilization Siderophore 3.3. Characterization of Rhizobacteria PGP Potential. The Ammonia Protease plant microbiome is a key determinant of plant health and IAA Cellulase productivity. Plant-associated microbes can help plants stim- (b) ulate growth, promote biotic and abiotic stress resistance and 100 influence crop yield and quality by nutrient mobilization and transport [6]. While the possibility to contribute to control 80 biotic stresses by plant-associated microorganisms is well characterized, less is known for abiotic stress. However, 60 several promising examples of stress protecting bacteria are 40 already reported in the literature [7, 38, 53]. Recent works (%) Isolates demonstrated that drought-exposed plants cultivated under 20 desert farming are colonized by bacterial communities with 0 high PGP potential [7, 8]. Such a PGP potential can promote BD-1 BD-5 BD-8 BD-9 BD-16 BD-B BD-C increased tolerance to water shortage, mediated by the induc- ∘ tion of a larger root system (up to 40%) that enhances water PEG 30% T≥45C uptake [7]. To assess if the oasis date palm PGP potential was pH ≤4 NaCl ≥10 conserved in the rhizosphere soil, 98 isolates were evaluated pH ≥10 for a series of PGP traits. The majority (85%) of isolates (c) showed multiple PGP activities, which may promote plant Figure 2: Diversity and functionality of cultivable bacteria islated growth directly, indirectly, or synergistically. Only 15% of from date palm rhizosphere. (a) Phylogenetic identification at the the rhizobacteria showed one or no activity, while no strains genus level of culturable bacteria associated with date palm rhizo- displayed all the screened PGP activities. The most common sphere. (b) Percentage of date palm rhizosphere-associated bacteria PGP trait was auxin production (83%), followed by ammonia showing PGP activity. (c) Percentage of isolates displaying the synthesis (63%) and biofertilization activities such as solu- assayed abiotic stress tolerance in the bacterial collection of strains bilization of phosphates (48%) and siderophore production associated with date palm cultivated in the seven oases analysed. BioMed Research International 7

(44%). In our rhizobacterial collection, the IAA production The tested rhizobacteria could grow in a wide pH range. wasequallydistributedamongthesevenoasesselected Within the bacterial collection 96% and the 75% of the strains along the aridity transect (Figure 2(b)), similarly to previous were facultative alkalophiles able to grow in basic media (up observations in other arboreal plants cultivated along a to pH = 12), while 34% of them could grow in acidic media latitude transect [11], confirming that IAA synthesis is a (pH = 4) and only 6% was facultative acidophiles growing widespread PGP trait. The IAA production ranged from 2.5 down to pH = 3. to 85 𝜇g/mL with 49% of the strains producing an amount 𝜇 ranging from 10 to 20 g/mL and the 38% showing higher 4. Conclusion levels of IAA (more than 20 𝜇g/mL). As already described in the literature, Pseudomonas, Bacillus, Pantoea, Staphylo- Date palm represents the key plant species in desert oases coccus,andMicrobacterium were the most abundant taxa being essential in determining the oasis microclimate that can implicated in IAA production [48, 54, 55]. The high frequency allow agriculture. Palm exerts both physical and functional of IAA producing strains suggests a role of PGP bacteria services involved in the creation of ideal condition for desert in contributing to regulate the root surface extension and farming. Palm root system and rhizosphere soil showed a consequently the potential of water and nutrient uptake [56]. complex diversity that enclosed a reservoir of PGP bacteria Phosphorus, together with iron and nitrogen, is a key involved in the regulation of plant homeostasis. Future nutrient for plant, particularly in oasis soil where the availability of nutrient sources of animal origin is scarce [57]. work is needed to perform experiment about the ability of The ability of rhizobacteria to solubilize phosphate (48%) selected bacterial isolates in promoting plant growth under through the production of organic acids or phytases can be greenhouse and field conditions. In this context, the selection very important in arid ecosystems [58, 59]. Strains of Pantoea, of autochthonous bacteria, together with the desert farming Enterobacter, Pseudomonas, Streptomyces,andRhizobium practices, could have promising perspectives for sustainable genera were the most efficient solubilizers, as previously agriculture in oasis ecosystem. showedinotheraridcontextssuchasTunisiangrapevine [48]anddifferentcropsinBolivia[60]. Several siderophore- Conflict of Interests producing bacteria were observed in the rhizosphere (44%) probably because this PGP trait confers competitive colo- The authors declare that there is no conflict of interests nization ability in iron-limiting soils. Iron is made available regarding the publication of this paper. fortheplanthostandconsequentlyexertsabiocontrolrole reducing iron-dependent spore germination of fungi [61]. Authors’ Contribution The siderophore-producing bacteria belonged mainly to the Pseudomonas genus (67%), followed by Bacillus (7%) and Raoudha Ferjani, Ramona Marasco, and Eleonora Rolli Pantoea (7%). Predominance of siderophore release by Pseu- contributed equally to the work. domonas bacteria was already reported in the rhizosphere of other plants [62, 63]. In addition to siderophore production, cell wall degrading enzymes implicated in fungal inhibition Acknowledgments and the organic matter turnover [51] were investigated. The 49% and 15% of the examined isolates were able to produce This work was supported by the project BIODESERT GA- proteases and cellulases, respectively, with the most active 245746 “Biotechnology from desert microbial extremophiles strains belonging to Serratia marcescens and Sinorhizobium for supporting agriculture research potential in Tunisia and Southern Europe” (European Union), Fondazione Project meliloti,respectively[64, 65]. Ammonia production can indi- ∘ rectly affect plant growth through nitrogen supply [66]. This BIOGESTECA n 15083/RCC “Fondo per la promozione trait was represented in 64% of the isolates, confirming its di accordi istituzionali” (Regione Lombardia, Italy) through spread in the palm-bacteria association. a fellowship to RM. ER was supported by Universita` degli Further analyses were performed to evaluate the adapt- Studi di Milano, DeFENS, European Social Fund (FSE), and ability of isolates to abiotic stresses (Figure 2(c)). Drought Regione Lombardia (contract “Dote Ricerca”). Thanks are stress resistance was presented by 95% of the strains that due to Marco Fusi for invaluable help in statistical analysis. could grow in presence of increasing concentrations of PEG. Research reported in this publication was supported by ∘ Mostofthestrains(98%)wereabletogrowat45C, while theKingAbdullahUniversityofScienceandTechnology ∘ only 39% at 50 C. The capacity to tolerate high temperature (KAUST). ∘ drastically decreased (5%) at 55 CandonlyBacillus and Pseudomonas strains showed this ability [67, 68]. Moderate References halotolerance was presented by 75% of the isolates, while 50% tolerated up to 15% NaCl, 20% actively grew in presence of [1] C. T. Chao and R. R. Krueger, “The date palm (Phoenix dactylif- 20% NaCl, and only the 6% were extremely halotolerant (25% era L.): overview of biology, uses, and cultivation,” HortScience, NaCl), indicating salinity as a major selective factor for the vol.42,no.5,pp.1077–1082,2007. bacterial microbiomes in the Tunisian date palm oases. The [2] H. de Haas, Migration and Agricultural Transformations in the formulation of halotolerant PGPR could be an interesting Oases of Morocco and Tunisia,KNAG,Utrecht,TheNether- alternative for agriculture productivity in the oasis [69]. lands, 2001. 8 BioMed Research International

[3]M.A.Elhoumaizi,M.Saaidi,A.Oihabi,andC.Cilas,“Pheno- [18] S.F.Altschul,W.Gish,W.Miller,E.W.Myers,andD.J.Lipman, typic diversity of date-palm cultivars (Phoenix dactylifera L.) “Basic local alignment search tool,” Journal of Molecular Biology, from Morocco,” Genetic Resources and Crop Evolution,vol.49, vol. 215, no. 3, pp. 403–410, 1990. no. 5, pp. 483–490, 2002. [19] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and [4]H.Hammadi,R.Mokhtar,E.Mokhtar,andF.Ali,“New S. Kumar, “MEGA5: molecular evolutionary genetics analysis approach for the morphological identification of date palm using maximum likelihood, evolutionary distance, and max- (Phoenix dactylifera L.) cultivars from Tunisia,” Pakistan Journal imum parsimony methods,” Molecular Biology and Evolution, of Botany,vol.41,no.6,pp.2671–2681,2009. vol.28,no.10,pp.2731–2739,2011. [5] Z. Sen, A. Al Alsheikh, A. M. Al-Dakheel et al., “Climate change [20] N. Saitou and M. Nei, “The neighbor-joining method: a new and Water Harvesting possibilities in arid regions,” Interna- method for reconstructing phylogenetic trees,” Molecular Biol- tional Journal of Global Warming,vol.3,no.4,pp.355–371,2011. ogy and Evolution,vol.4,no.4,pp.406–425,1987. [6]C.M.Ryu,M.A.Farag,C.H.Huetal.,“Bacterialvolatilespro- [21] H. Ouzari, A. Khsairi, N. Raddadi et al., “Diversity of auxin- mote growth in Arabidopsis,” Proceedings of the National Acad- producing bacteria associated to Pseudomonas savastanoi- emy of Sciences of the United States of America,vol.100,no.8, induced olive knots,” JournalofBasicMicrobiology,vol.48,no. pp. 4927–4932, 2013. 5, pp. 370–377, 2008. [7] A. de Zelicourt, M. Al-Yousif, and H. Hirt, “Rhizosphere [22] C. S. Nautiyal, “An efficient microbiological growth medium microbes as essential partners for plant stress tolerance,” Molec- for screening phosphate solubilizing microorganisms,” FEMS ular Plant,vol.6,no.2,pp.242–245,2013. Microbiology Letters,vol.170,no.1,pp.265–270,1999. [8] R. Marasco, E. Rolli, B. Ettoumi et al., “A drought resistance- [23] S. Perez-Miranda,N.Cabirol,R.George-T´ ellez,´ L. S. Zamudio- promoting microbiome is selected by root system under desert Rivera, and F. J. Fernandez,´ “O-CAS, a fast and universal farming,” PLoS ONE,vol.7,no.10,ArticleIDe48479,2012. method for siderophore detection,” Journal of Microbiological [9] M. Koberl,¨ H. Muller,¨ E. M. Ramadan, and G. Berg, “Desert Methods,vol.70,no.1,pp.127–131,2007. farming benefits from microbial potential in arid soils and [24] J. C. Cappuccino and N. Sherman, Microbiology: A Laboratory promotes diversity and plant health,” PLoS ONE,vol.6,no.9, Manual, Benjamin Cummings, New York, NY, USA, 3rd edi- Article ID e24452, 2011. tion, 1992. [10] K. Kumar, N. Amaresan, S. Bhagat, K. Madhuri, and R. C. Sriv- astava, “Isolation and characterization of rhizobacteria associ- [25] M. E. Cattelan, P. G. Hartel, and J. J. Fuhrmann, “Screening for ated with coastal agricultural ecosystem of rhizosphere soils of plant growth-promoting rhizobacteria to promote early soy- cultivated vegetable crops,” World Journal of Microbiology and bean growth,” Soil Science Society of America Journal,vol.63, Biotechnology,vol.27,no.7,pp.1625–1632,2011. no. 6, pp. 1670–1680, 1999. [11] R. Marasco, E. Rolli, M. Fusi et al., “Plant growth promotion [26] M. J. Anderson, “A new method for non-parametric multivari- potential is equally represented in diverse grapevine root- ate analysis of variance,” Austral Ecology,vol.26,no.1,pp.32– associated bacterial communities from different biopedocli- 46, 2001. matic environments,” BioMed Research International,vol.2013, [27] M. J. Anderson, DISTLM v.2: A FORTRAN Computer Program Article ID 491091, 17 pages, 2013. to Calculate a Distance-Based Multivariate Analysis for a Linear [12] A. A. Othman, W. M. Amer, M. Fayez, and N. A. Hegazi, “Rhi- Model, Department of Statistics, University of Auckland, 2002. zosheath of sinai desert plants is a potential repository for [28] H. Bozdogan, “Model selection and Akaike’s Information Cri- associative diazotrophs,” Microbiological Research,vol.159,no. terion (AIC): the general theory and its analytical extensions,” 3, pp. 285–293, 2004. Psychometrika. A Journal of Quantitative Psychology,vol.52,no. [13] V. Saul-Tcherkas, A. Unc, and Y. Steinberger, “Soil microbial 3,pp.345–370,1987. diversity in the vicinity of desert shrubs,” Microbial Ecology,vol. [29] K. Clarke and R. Gorley, “PRIMER v6.” User Manual/Tutorial, 65,no.3,pp.689–699,2013. Plymouth Routine in Multivariate Ecological Research,Plymouth [14] G. Muyzer, E. C. de Waal, and A. G. Uitterlinden, “Profiling Marine Laboratory, 2006. of complex microbial populations by denaturing gradient gel [30]M.J.Anderson,R.N.Gorley,andK.R.Clarke,PERMAN- electrophoresis analysis of polymerase chain reaction-amplified OVA+ for PRIMER: Guide to Software and Statistical Methods, genes coding for 16S rRNA,” Applied and Environmental Micro- PRIMER-E, Plymouth, UK, 2008. biology,vol.59,no.3,pp.695–700,1993. [31]W.M.Cook,K.T.Lane,B.L.Foster,andR.D.Holt,“Island [15]V.C.Sheffield,D.R.Cox,L.S.Lerman,andR.M.Myers, theory, matrix effects and species richness patterns in habitat “Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) fragments,” Ecology Letters,vol.5,no.5,pp.619–623,2002. to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes,” Proceed- [32] T. S. Walker, H. P. Bais, E. Grotewold, and J. M. Vivanco, “Root ings of the National Academy of Sciences of the United States of exudation and rhizosphere biology,” Plant Physiology,vol.132, America,vol.86,no.1,pp.232–236,1989. no. 1, pp. 44–51, 2003. [16] G. Merlino, A. Rizzi, A. Schievano et al., “Microbial commu- [33] R. Angel, M. I. M. Soares, E. D. Ungar, and O. Gillor, “Biogeog- nity structure and dynamics in two-stage vs single-stage ther- raphy of soil archaea and bacteria along a steep precipitation mophilic anaerobic digestion of mixed swine slurry and market gradient,” ISME Journal,vol.4,no.4,pp.553–563,2010. bio-waste,” Water Research,vol.47,no.6,pp.1983–1995,2013. [34]A.Bachar,M.I.M.Soares,andO.Gillor,“Theeffectofresource [17] I. Fhoula, A. Najjari, Y. Turki, S. Jaballah, A. Boudabous, and islands on abundance and diversity of bacteria in arid soils,” H. Ouzari, “Diversity and antimicrobial properties of lactic acid Microbial Ecology,vol.63,no.3,pp.694–700,2012. bacteria isolated from rhizosphere of olive trees and desert [35]S.C.Cary,I.R.McDonald,J.E.Barrett,andD.A.Cowan, truffles of tunisia,” BioMed Research International,vol.2013, “On the rocks: The microbiology of Antarctic Dry Valley soils,” Article ID 405708, 14 pages, 2013. Nature Reviews Microbiology,vol.8,no.2,pp.129–138,2010. BioMed Research International 9

[36] T.Caruso, Y.Chan, D. C. Lacap, M. C. Y.Lau, C. P.McKay, and S. [50]D.Muleta,F.Assefa,K.Hjort,S.Roos,andU.Granhall,“Char- B. Pointing, “Stochastic and deterministic processes interact in acterization of rhizobacteria isolated from wild Coffea arabica the assembly of desert microbial communities on a global scale,” L,” Engineering in Life Sciences,vol.9,no.2,pp.100–108,2009. ISME Journal,vol.5,no.9,pp.1406–1413,2011. [51] G. Kumar, N. Kanaujia, and A. Bafana, “Functional and phylo- [37] M. Koberl,¨ E. M. Ramadan, M. Adam et al., “Bacillus and Strep- genetic diversity of root-associated bacteria of Ajuga bracteosa tomyces were selected as broad-spectrum antagonists against in Kangra valley,” Microbiological Research, vol. 167, no. 4, pp. soilborne pathogens from arid areas in egypt,” FEMS Microbi- 220–225, 2012. ology Letters,vol.342,no.2,pp.168–178,2013. [52] D. El Hidri, A. Guesmi, A. Najjari et al., “Cultivation-dependant [38] E. Armada, A. Roldan,´ and R. Azcon, “Differential activity of assessment, diversity, and ecology of haloalkaliphilic bacteria autochthonous bacteria in controlling drought stress in native in arid saline systems of southern Tunisia,” BioMed Research lavandula and salvia plants species under drought conditions in International, vol. 2013, Article ID 648141, 15 pages, 2013. natural arid soil,” Microbial Ecology,vol.67,no.2,pp.410–420, [53] P. Alavi, M. R. Starcher, C. Zachow, H. Muller,¨ and G. Berg, 2014. “Root-microbe systems: the effect and mode of interaction [39]A.Bachar,A.Al-Ashhab,M.I.M.Soaresetal.,“Soilmicrobial of stress protecting agent (SPA) Stenotrophomonas rhizophila abundance and diversity along a low precipitation gradient,” DSM14405T,” Frontiers in Plant Science,vol.4,p.141,2013. Microbial Ecology,vol.60,no.2,pp.453–461,2010. [54] O. Barazani and J. Friedman, “Is IAA the major root growth factor secreted from plant-growth-mediating bacteria?” Journal [40] H. Bertrand, R. Nalin, R. Bally, and J.-C. Cleyet-Marel, “Iso- of Chemical Ecology,vol.25,no.10,pp.2397–2406,1999. lation and identification of the most efficient plant growth- promoting bacteria associated with canola (Brassica napus),” [55] P. Trivedi, T. Spann, and N. Wang, “Isolation and character- Biology and Fertility of Soils,vol.33,no.2,pp.152–156,2001. ization of beneficial bacteria associated with citrus roots in Florida,” Microbial Ecology,vol.62,no.2,pp.324–336,2011. [41] M. A. Siddikee, P. S. Chauhan, R. Anandham, G.-H. Han, and T. Sa, “Isolation, characterization, and use for plant growth pro- [56] R. Marasco, E. Rolli, G. Vigani et al., “Are drought-resistance motion under salt stress, of ACC deaminase-producing halotol- promoting bacteria cross-compatible with different plant mod- erant bacteria derived from coastal soil,” Journal of Microbiology els?” Plant Signaling and Behavior,vol.8,no.10,ArticleID and Biotechnology,vol.20,no.11,pp.1577–1584,2010. e26741, 2013. [57]D.P.Schachtman,R.J.Reid,andS.M.Ayling,“Phosphorus [42] F. Mapelli, R. Marasco, E. Rolli et al., “Potential for plant growth uptake by plants: from soil to cell,” Plant Physiology,vol.116,no. promotion of rhizobacteria associated with Salicornia growing 2,pp.447–453,1998. in Tunisian hypersaline soils,” BioMed Research International, vol.2013,ArticleID248078,13pages,2013. [58] P. Vyas, P. Rahi, and A. Gulati, “Stress tolerance and genetic variability of phosphate-solubilizing fluorescent Pseudomonas [43] D. Daffonchio, A. De Biase, A. Rizzi, and C. Sorlini, “Interspe- from the cold deserts of the trans-himalayas,” Microbial Ecology, cific, intraspecific and interoperonic variability in the 16S rRNA vol.58,no.2,pp.425–434,2009. gene of methanogens revealed by length and single-strand con- formation polymorphism analysis,” FEMS Microbiology Letters, [59] K. W.Ndung’u-Magiroi, L. Herrmann, J. R. Okalebo, C. O. Oth- vol. 164, no. 2, pp. 403–410, 1998. ieno, P. Pypers, and D. Lesueur, “Occurrence and genetic diversity of phosphate-solubilizing bacteria in soils of differing [44] D. Daffonchio, S. Borin, G. Frova, P. L. Manachini, and C. chemical characteristics in Kenya,” Annals of Microbiology,vol. Sorlini, “PCR fingerprinting of whole genomes: the spacers 62,no.3,pp.897–904,2012. between the 16s and 23S rRNA genes and of intergenic tRNA [60] M. Furnkranz,¨ H. Muller,¨ and G. Berg, “Characterization of gene regions reveal a different intraspecific genomic variability plant growth promoting bacteria from crops in Bolivia,” Journal of Bacillus cereus and Bacillus licheniformis,” International Jour- of Plant Diseases and Protection,vol.116,no.4,pp.149–155,2009. nal of Systematic Bacteriology, vol. 48, no. 1, pp. 107–116, 1998. [61] C. M. Ribeiro and E. J. B. N. Cardoso, “Isolation, selection and [45] D. Daffonchio, A. Cherif, and S. Borin, “Homoduplex and het- characterization of root-associated growth promoting bacte- eroduplex polymorphisms of the amplified ribosomal 16S-23S riainBrazilPine(Araucaria angustifolia),” Microbiological internal transcribed spacers describe genetic relationships in Research,vol.167,no.2,pp.69–78,2012. the “Bacillus cereus group”,” AppliedandEnvironmentalMicro- biology,vol.66,no.12,pp.5460–5468,2000. [62] F. Tian, Y. Ding, H. Zhu, L. Yao, and B. Du, “Genetic diversity of siderophore-producing Bacteria of tobacco rhizosphere,” [46] V. Gurtler¨ and V. A. Stanisich, “New approaches to typing Brazilian Journal of Microbiology,vol.40,no.2,pp.276–284, and identification of bacteria using the 16S-23S rDNA spacer 2009. region,” Microbiology,vol.142,no.1,pp.3–16,1996. [63] A. Sharma and B. N. Johri, “Growth promoting influence of [47] G. Berg and K. Smalla, “Plant species and soil type cooperatively siderophore-producing Pseudomonas strains GRP3A and PRS9 shape the structure and function of microbial communities in in maize (Zea mays L.) under iron limiting conditions,” Micro- the rhizosphere,” FEMS Microbiology Ecology,vol.68,no.1,pp. biological Research,vol.158,no.3,pp.243–248,2003. 1–13, 2009. [64] D. J. M. Kumar, L. Lawrence, R. Rajan, S. Priyadarshini, S. Yach- [48] F. Garcia-Pichel, S. L. Johnson, D. Youngkin, and J. Belnap, ittybabu, and P. T. Kalaichelvan, “Characterization of lipase and “Small-scale vertical distribution of bacterial biomass and protease from Serratia marcescens DEPTK21 and its destaining diversity in biological soil crusts from arid lands in the Colorado capability,” Asian Journal of Experimental Biological Sciences, Plateau,” Microbial Ecology,vol.46,no.3,pp.312–321,2003. vol. 3, no. 3, pp. 621–628, 2012. [49] P.Joshi and A. B. Bhatt, “Diversity and function of plant growth [65] P. Michaud, A. Belaich, B. Courtois, and J. Courtois, “Cloning, promoting Rhizobacteria associated with wheat Rhizosphere in sequencing and overexpression of a Sinorhizobium meliloti North Himalayan Region,” International Journal of Environmen- M5N1CS carboxymethyl-cellulase gene,” Applied Microbiology tal Sciences, vol. 1, no. 6, p. 1135, 2011. and Biotechnology,vol.58,no.6,pp.767–771,2002. 10 BioMed Research International

[66]P.A.Wani,M.S.Khan,andA.Zaidi,“Synergisticeffectsofthe inoculation with nitrogen-fixing and phosphate-solubilizing rhizobacteria on the performance of field-grown chickpea,” Journal of Plant Nutrition and Soil Science,vol.170,no.2,pp. 283–287, 2007. [67] P. Scheldeman, L. Herman, S. Foster, and M. Heyndrickx, “Bacillus sporothermodurans and other highly heat-resistant spore formers in milk,” JournalofAppliedMicrobiology,vol.101, no. 3, pp. 542–555, 2006. [68] S. S. Kanwar, M. Gupta, R. Gupta, R. K. Kaushal, and S. S. Chimni, “Properties of hydrogel-entrapped lipase of thermophilic Pseudomonas aeruginosa BTS-2,” Indian Journal of Bio- technology,vol.5,no.3,pp.292–297,2006. [69] I. L. D. Diatta, A. Kane, C. E. Agbangba et al., “Inoculation with arbuscular mycorrhizal fungi improves seedlings growth of two sahelian date palm cultivars (Phoenix dactylifera L., cv. Nakhla hamra and cv. Tijib) under salinity stresses,” Advances in Bio- science and Biotechnology,vol.5,no.1,pp.64–72,2014. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 296472, 9 pages http://dx.doi.org/10.1155/2014/296472

Research Article Pentachlorophenol Degradation by Janibacter sp., a New Actinobacterium Isolated from Saline Sediment of Arid Land

Amel Khessairi,1,2 Imene Fhoula,1 Atef Jaouani,1 Yousra Turki,2 Ameur Cherif,3 Abdellatif Boudabous,1 Abdennaceur Hassen,2 and Hadda Ouzari1

1 UniversiteTunisElManar,Facult´ e´ des Sciences de Tunis (FST), LR03ES03 Laboratoire de Microorganisme et Biomolecules´ Actives, Campus Universitaire, 2092 Tunis, Tunisia 2 Laboratoire de Traitement et Recyclage des Eaux, Centre des Recherches et Technologie des Eaux (CERTE), Technopoleˆ Borj-Cedria,´ B.P. 273, 8020 Soliman, Tunisia 3 Universite´ de Manouba, Institut Superieur´ de Biotechnologie de Sidi Thabet, LR11ES31 Laboratoire de Biotechnologie et Valorization des Bio-Geo Resources, Biotechpole de Sidi Thabet, 2020 Ariana, Tunisia

Correspondence should be addressed to Hadda Ouzari; [email protected]

Received 1 May 2014; Accepted 17 August 2014; Published 17 September 2014

Academic Editor: George Tsiamis

Copyright © 2014 Amel Khessairi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Many pentachlorophenol- (PCP-) contaminated environments are characterized by low or elevated temperatures, acidic or alkaline pH, and high salt concentrations. PCP-degrading microorganisms, adapted to grow and prosper in these environments, play an important role in the biological treatment of polluted extreme habitats. A PCP-degrading bacterium was isolated and characterized from arid and saline soil in southern Tunisia and was enriched in mineral salts medium supplemented with PCP as source of carbon and energy. Based on 16S rRNA coding gene sequence analysis, the strain FAS23 was identified as Janibacter sp. As revealed by high performance liquid chromatography (HPLC) analysis, FAS23 strain was found to be efficient for PCP removal in the presence of1% of glucose. The conditions of growth and PCP removal by FAS23 strain were found to be optimal in neutral pH and at a temperature ∘ of 30 C. Moreover, this strain was found to be halotolerant at a range of 1–10% of NaCl and able to degrade PCP at a concentration up to 300 mg/L, while the addition of nonionic surfactant (Tween 80) enhanced the PCP removal capacity.

1. Introduction environment [7]. Although contamination of soils and waters with chemically synthesized PCP is a serious environmental The polyorganochlorophenolic (POP) compounds have been problem, their remediation may be possible using physi- extensively used as wide spectrum biocides in industry cal, chemical, and biological methods [8]. Bioremediation and agriculture [1]. The toxicity of these compounds tends represents a choice process, thanks to its low costs and to increase according to their degree of chlorination [2]. reduction of toxic residue generated in the environment. The Among chlorinated phenols, pentachlorophenol (PCP) has biodegradation of PCP has been studied in both aerobic and been widely used as wood and leather preservative, owing anaerobic systems. Aerobic degradation of PCP especially to its toxicity toward bacteria, mould, algae, and fungi [3]. has been extensively studied and several bacterial isolates However, PCP is also toxic to all forms of life since it is were found to degrade and use PCP as a sole source of an inhibitor of oxidative phosphorylation [4]. The extensive carbon and energy. The most studied aerobic PCP-degrading exposure to PCP could cause cancer, acute pancreatitis, microorganisms included Mycobacterium chlorophenolicum immunodeficiency, and neurological disorders [5]. Conse- [9], Alcaligenes sp. [10], Rhodococcus chlorophenolicus [11], quently, this compound is listed among the priority pollutants Flavobacterium [12], Novosphingobium lentum [13]andSph- of the US Environmental Protection Agency [6]. Moreover, it ingomonas chlorophenolica [14], Bacillus [15], Pseudomonas is recalcitrant to degradation because of its stable aromatic [16], and Acinetobacter [17], as well as some fungi species. ring and high chloride contents, thus persisting in the Saline and arid environments are found in a wide variety 2 BioMed Research International of aquatic and terrestrial ecosystems. A low taxonomic wasspreadonthesurfaceofyeastextract-mannitolmedium biodiversity is observed in all these saline environments [18], (YEM). YEM medium contained the following components most probably due to the high salt concentrations prevailing at the specified concentrations (in g/L): mannitol, 5; yeast in these environments. Moreover, the biodegradation process extract, 0.5; MgSO4⋅7H2O, 0.2; NaCl, 0.1; K2HPO4,0.5;Na is difficult to perform under saline conditions [19]. Besides gluconate, 5; agar, 15; pH = 6, 8. After sterilization for 20 min ∘ these metabolical and physiological features, halophilic and at 120 C, 1 mL of 16.6% CaCl2 solution was added to 1 liter halotolerant microorganisms are known to play important of YEM medium (1 : 1000). The plates were then incubated at ∘ roles in transforming and degrading waste and organic pol- 30 C for 7 days. Pure cultures of the isolates were obtained by lutants in saline and arid environment [20]. These microor- streaking a single colony on the same medium. ganisms, particularly actinobacteria, are frequently isolated from extreme environments such as Sabkha, Chott, and 2.3. 16S rRNA Gene Amplification and Sequence Analysis. Sahara which are known to have a great metabolic diversity For DNA extraction, the FAS23 strain was grown in tryptic and biotechnological potential. The occurrence of actinobac- soy broth (TSB) containing (in g/L) casein peptone, 17; soya teria in saline environment and their tolerance to high peptone, 3; glucose, 2.5; sodium chloride, 5; dipotassium salt concentrations were thus described [21]. However, few hydrogen phosphate, 4. DNA extraction was performed actinobacteria genera, such as Arthrobacter [22]andKocuria using CTAB/NaCl method as described by Wilson [37]and [23], were reported for PCP-degradation process. The genus modified by using lysozyme (1 mg/mL) for cell wall digestion. Janibacter which is recognized by Martin et al. [24]belongsto The 16S rRNA gene was amplified using universal primers 󸀠 the family Intrasporangiaceae in the Actinomycetales order SD-Bact-0008-a-S-20 (5 -AGA GTT TGA TCC TGG CTC 󸀠 󸀠 and included five major species, J. limosus [24], J. terrae AG- 3) and S-D-Bact-1495-a-A-20 (5 -CTA CGG CTA CCT 󸀠 [25], J. melonis [26], J. corallicola [27], and J. anophelis [28]. TGT TAC GA- 3) [38]. PCR was performed in a final volume Interestingly, most of these species were reported for their of 25 𝜇L containing 1 𝜇L of the template DNA; 0.5 𝜇M of each ability to degrade a large spectrum of aromatic and/or chlori- primer; 0.5 𝜇M of deoxynucleotide triphosphate (dNTP); nated compounds including polychlorinated biphenyls [29], 2.5 𝜇L 10X PCR buffer for Taq polymerase; MgCl2 1.5 mM; monochlorinated dibenzo-p-dioxin [30], dibenzofuran [31], 1 UI of Taq polymerase. The amplification cycle was as ∘ anthracene, phenanthrene [32], dibenzo-p-dioxin, carbazole, follows: denaturation step at 94 Cfor3min,followedby35 ∘ ∘ ∘ diphenyl ether, fluorene33 [ ], and polycyclic aromatic hydro- cycles (45 sec at 94 C, 1 min at 55 C,and2minat72C) plus ∘ carbons [34]. However, no data reporting PCP degradation one additional cycle at 72 C for 7 min as a final elongation by Janibacter members was described. PCP and other POP step. The 16S rDNA PCR amplicons were purified with compounds shared many physical properties, which limited Exonuclease-I and Shrimp Alkaline Phosphatase (Exo-Sap, biodegradation processes, and one of these properties was Fermentas, Life Sciences) following the manufacturer’s stan- their lower solubility and therefore low bioavailability to the dardprotocol.SequenceanalysesofthepurifiedDNAswere degrading bacteria. Nevertheless, the use of surfactants such performed using a Big Dye Terminator cycle sequencing kit as Tween 80 has the potential to increase the biodegradation V3.1 (Applied Biosystems) and an Applied Biosystems 3130XL rates of hydrophobic organic compounds by increasing the Capillary DNA Sequencer machine. Sequence similarities total aqueous solubility of these pesticides [35]. In this study, were found by BLAST analysis [39]usingtheGenBankDNA we evaluated for the first time the PCP removal potential, databases (http://www.ncbi.nlm.nih.gov/) and the Riboso- under different physicochemical conditions, by Janibacter sp., mal Database Project (RDP). Phylogenetic analyses of the 16S a halotolerant actinobacterium member isolated from arid rRNA gene sequences were conducted with Molecular Evolu- andsalinelandinsouthernTunisia. tionary Genetics Analysis (MEGA) software, version 5 [40]. Trees were constructed by using neighbor-joining method 2. Materials and Methods [41]. The sequence was deposited in GenBank database under the accession number KC959984. 2.1. Chemicals and Solvents. PCP (MW 266.34 and >99% purity) and acetonitrile (HPLC grade) were purchased from 2.4. Degradation of PCP by Isolated Strain. The kinetics of Sigma Aldrich (USA). All other inorganic chemicals used to the PCP removal under different conditions were conducted prepare the different media are commercially available with in 500 mL flasks, sealed with cotton stoppers, containing highestpurityandareusedwithoutfurtherpurification. 100 mL of mineral salt medium (MSM) adjusted to pH 6.9, supplemented with 1% glucose and inoculated with 6 2.2. Sample Collection and PCP-Degrading Bacterium Isola- 1% of 10 CFU/mLofthestrainFAS23.TheMSMcon- tion. The sediment samples were collected in March 2011 tained the following components at the specified concen- from arid and saline ecosystems belonging to the site “Sebkha trations (in g/L): KH2PO4,0.8;Na2HP4,0.8;MgSO4⋅7H2O, ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 El Naouel” with GPS coordinates: N 34 26 951 E0954 102 0.2; CaCl2⋅2H2O, 0.01; NH4Cl, 0.5, plus 1 mL of trace altitude 150 ft/46 m, in southern Tunisia. Bacterial isolation metal solution which includes (in mg/L) FeSO4⋅7H2O, 5; was performed as described by Rosch¨ et al. [36]withsome ZnSO4⋅7H2O, 4; MnSO4⋅4H2O, 0.2; NiCl⋅6H2O, 0.1; H3BO3, modifications: 10 g of soil sample was suspended in 100 mL of 0.15; CoCl2⋅6H2O, 0.5; ZnCl2 0.25;andEDTA,2.5.PCP phosphate-buffered salt solution (137 mM NaCl, 2.7 mM KCl, was added to the medium after autoclaving [19]. When 10 mM Na2HPO4,and2mMKH2PO4) and stirred vigorously necessary, solid MSM plates were prepared by adding 15 g/L for 30 min. The soil suspension was diluted and 0.1 mL sample bacteriological grade agar. The inoculum was prepared as BioMed Research International 3

Janibacter melonis (JX865444) Janibacter marinus (AY533561) 40 Janibacter terrae (KC469957) 52 Janibacter hoylei (FR749912) Janibacter sanguinis (JX435047) 58 Janibacter corallicola (NR 041558) 100 Janibacter sp. FAS23 82 Janibacter sp. ATCC 33790 (GU933618) Janibacter limosus (KC469951) 044984 76 Terrabacter tumescens (NR ) 99 Terracoccus luteus (NR 026412) Kocuria rhizophila (NR 026452) Arthrobacter chlorophenolicus (EU102284)

0.01

Figure 1: The phylogenetic position of Janibacter sp. strain in relation to some members of actinobacteria (genus of Janibacter, Sphingomonas, Terrabacter, Terracoccus, Kocuria, and Arthrobacter) based on 16S rRNA gene. Bootstrap values for a total of 1000 replicates are shown at the nodes of the tree. The scale bar corresponds to 0.05 units of the number of base substitutions per site changes per nucleotide. follows: overnight culture was centrifuged and the pellet was The morphological aspect of FAS23 strain culture on the rinsed twice with fresh MSM. PCP removal was monitored isolation medium YEM showed opaque, pale, cream, and during 144 h of incubation by varying different parameters: convex colonies with glistening surface. Cells were Gram- (i) initial pH; (ii) initial PCP concentrations: 20, 50, 100, positive, rod-shaped, and positive for catalase and oxidase 200, and 300 mg/L corresponding to 0.075 mM, 0.19 mM, tests. No growth under anaerobic conditions and no spore 0.37, 0.75 mM, and 1.14 mM, respectively; (iii) temperature formation were recorded. The optimal growth conditions of ∘ of incubation: 25, 30, and 37 C; (iv) NaCl concentrations: FAS23 strain were pH of 7.0–8.5 and a temperature range ∘ 10 g/L, 30 g/L, 60 g/L, and 100 g/L; (v) the addition of nonionic of 28–30 C. The strain was able to grow at a range of surfactant Tween 80 (40 mg/L). Bacterial cell growth was salt concentrations from 1 to 100 g/L of NaCl. 16S rDNA evaluated by measuring the optical density at 600 nm using sequencing and phylogenetic analysis allowed the assignment UV-VIS spectrophotometer (Spectro UVS-2700 Dual Beam of FAS23 strain to Janibacter sp. (Figure 1). Labomed, Inc) every 24 h of the incubation. Three controls were used: PCP-free MSM, uninoculated PCP containing 3.2. The Optimum Growth Conditions of Janibacter sp. Strain. MSM, and PCP containing MSM inoculated with heated The effect of physiological and biochemical variations (glu- inactivated cells. The cell suspension was centrifuged (5 min, cose supplement, temperature, pH, PCP concentration, and 8000 rpm) and the supernatant was filtered through 0.22 𝜇m presence of biosurfactant) on bacterial growth of Janibacter filters [16]. Samples of 100 𝜇L were applied to C18 reverse sp. FAS23 and PCP removal was studied. phase column (LiChrospher 100 RP-18 endcapped column, 250 mm × 4.6 mm i.d., and particle size of 5 𝜇m) at a flow −1 3.2.1. Effect of Glucose on the Growth of Janibacter sp. and PCP rate of 1 mL min . The retained molecules were eluted over Removal. The effect of glucose as cosubstrate on the growth 35 min using the following gradient: 1% (v/v) phosphoric of Janibacter sp. strain and PCP removal was studied in MSM. acid in water for 4 min, followed by an increase to 100% The result showed that the growth of the strain was possible (v/v) acetonitrile within 21 min which was kept constant for only after the addition of glucose (Figure 2(a)). As well, the 5 min and then decreased back to initial concentration and PCP was efficiently removed in the presence of glucose, and kept constant for another 5 min. PCP was quantified using 71.84% of PCP was degraded within 24 hours and more than external standards method. Percent removal was estimated 90% after 72 hFigure ( 2(b)). The obtained results indicated − using the following formula: removal (%) = area area/area the phenomenon of cometabolism in which microorganisms [42]. do not obtain energy from the transformation reaction; they rather require another substrate for growth [43]. 2.5. Statistical Analysis. Data were subjected to analysis of variance using SPSS software (version 14.0) and the mean 3.2.2. Effect of pH and Temperature on the Growth of the differences were compared by Student-Newman-Keuls com- Strain and PCP Removal. The effect of pH variations (4.0, parison test. A 𝑃 value of less than 0.05 was considered 6.9, and 9.0) on the growth and PCP removal was assessed statistically significant (test at 𝑃 < 0.05). Three replicates were (Figure 3). At both pH 4.0 and 9.0, a low rate of growth and prepared for each treatment. PCP removal was observed after 24 and 48 h of incubation. However, after 144 h of incubation, the rate of PCP removal 3. Results has reached values of 44.80% and 70.22% at pH 4.0 and pH 9.0, respectively. The optimum growth and PCP removal were 3.1. Isolation, Identification of FAS23 Strain, and 16S rDNA however observed at pH 6.9, as we noted a significant removal Sequence Based Phylogenetic Analysis. The bacterial strain of PCP of 71.84%, 84.47%, and 99.06% after 24, 48, and 144 h FAS23 was isolated from the saline and arid sediment. of incubation, respectively. The strain FAS23 was able to grow 4 BioMed Research International

2.0 100 1.8 1.6 80 1.4 nm 1.2 60 600 1.0 0.8 40 OD at 0.6 Residual PCP (%) Residual 0.4 20 0.2 0 0.0 0 24487296120144 0 24 48 72 96 120 144 Incubation period (h) Incubation period (h) + Glucose PCP Glucose PCP – Glucose PCP PCP (a) (b) Figure 2: The growth (a) and the PCP removal (b) in the presence and in deficiency of the supplementary carbon source (glucose: 1%)by Janibacter sp. FAS23. Error bars represent the standard deviation.

2.0 100 1.8 90 1.6 80 1.4 70

nm 1.2 60

600 1.0 50 0.8 40 OD at 0.6 30 Residual PCP (%) Residual 0.4 20 0.2 10 0.0 0 0 24487296120144 0 24 48 72 96 120 144 Incubation periods (h) Incubation period (h) pH 4-PCP pH 6.9 pH 4 pH 4 pH 9-PCP pH 6.9 pH 6.9-PCP pH 9 pH 9 (a) (b) ∘ Figure 3: (a) Growth of Janibacter sp. at different pH of culture medium: pH 4.0, pH 6.9, and pH 9.0 with 20 mg/L of PCP at30 C. (b) Effect of different pH on the PCP removal efficiency by Janibacter sp. Error bars represent the standard deviation.

∘ in the temperature range of 25–37 C, with an optimum at incubation time. Up to 100 mg/L, 50% of PCP could be ∘ ∘ 30 C. At 25 and 37 C, the growth of the bacterial strain, as removed after 72 h of incubation. However, with higher well as PCP removal, was affected (Figure 4). However, at concentrations (200 and 300 mg/L), equivalent level of PCP ∘ 25 C, the strain showed a better growth and PCP removal removal could be reached if the incubation time is extended. ∘ compared to temperature of 37 C. Likewise, the PCP removal ∘ was optimal at 30 Creaching71.84%and99.06%after24h 3.2.4.EffectofVariousNaClConcentrationsonthePCP and 144 h of incubation, respectively. Removal. The strain was tested for its ability to remove PCP (20 mg/L) at different NaCl concentrations (0%, 1%, 3%, 3.2.3. Effect of PCP Amount on the Growth and PCP Removal 6%, and 10%). The best rate of growth and PCP removal by Janibacter. Variation of PCP amount in the medium was recorded at 1% of NaCl (more than 92% after 144 h showed that the growth of the strain, as well as PCP removal, of incubation). The growth and thus the capacity of PCP decreased with the increase of PCP concentration (Figure 5). removal were inhibited when the concentration of sodium At low concentrations (20 and 50 mg/L), the bacterial strain chloride was increased (Figure 6). At 3% NaCl, the PCP was able to remove the majority of PCP after 72 h of removal was 72% after 144 h of incubation. When the NaCl BioMed Research International 5

2.0 100 1.8 90 1.6 80 1.4 70 1.2 nm 60

600 1.0 50 0.8 40 OD at

0.6 PCP (%) Residual 30 0.4 20 0.2 10

0.0 0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 Incubation period (h) Incubation period (h)

∘ ∘ ∘ 25 C-PCP 25 C 25 C ∘ ∘ ∘ 30 C-PCP 30 C 30 C ∘ ∘ ∘ 37 C-PCP 37 C 37 C (a) (b)

Figure 4: (a) Growth of Janibacter sp.atdifferenttemperaturesinpresenceof20mg/LofPCPandatpH6.9.(b)Effectoftemperaturechanges on the PCP removal efficiency by Janibacter sp. Error bars represent the standard deviation.

2.0 1.8 100 1.6 90 1.4 80 70

nm 1.2 60

600 1.0 50 0.8

OD at 40 0.6 Residual PCP (%) Residual 30 0.4 20 0.2 10 0.0 0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 Incubation period (h) Incubation period (h)

0 100 20 200 20 200 50 300 50 300 100 (a) (b) Figure 5: (a) Growth of Janibacter sp. in the presence of 0, 20, 50, 100, 200, and 300 mg/L of PCP. (b) Effect of different PCP concentrations on the PCP removal by Janibacter sp. Error bars represent the standard deviation. concentration was increased to 6% and 10%, PCP removal of PCP (300 mg/mL) was improved by 30% after 72 h of falls to 46.53% and 17.62%, respectively. incubation, compared to the control (Figure 7(b)).

3.2.5.EffectofNonionicSurfactantTween80ontheBiodegra- 4. Discussion dation of PCP. In this study, the nonionic surfactant Tween 80 was found to enhance the growth and PCP-biodegradation The strain FAS23 isolated from saline sediment collected process (Figure 7). Interestingly, removal of high amount from Tunisian arid ecosystems was identified as an 6 BioMed Research International

2.0 100 1.8 90 1.6 80 1.4 70 nm 1.2 60

600 1.0 50 0.8 40 OD at 0.6 30 Residual PCP (%) Residual 0.4 20 0.2 10 0.0 0 0 24487296120144 0 24 48 72 96 120 144 Incubation period (hours) Incubation period (hours)

0% 6% 0% 6% 1% 10% 1% 10% 3% 3% (a) (b) Figure 6: (a) Growth of Janibacter sp. in MS medium supplemented with different concentrations of NaCl: 0%, 1%, 3%, 6%, and 10% in the ∘ presence of 20 mg/L of PCP at 30 C. (b) Effect of NaCl on the PCP removal efficiency by Janibacter sp. Error bars represent the standard deviation.

2.0 100 1.8 90 1.6 80 1.4 70 nm 1.2 60 600 1.0 50 0.8 40 OD at 0.6 30 Residual PCP (%) Residual 0.4 20 0.2 10 0.0 0 0 24 48 72 96 120 144 0 24 48 72 96 120 144 Incubation period (h) Incubation period (h)

20-T80 300-T80 20-T80 300-T80 20 300 20 300 (a) (b)

Figure 7: (a) Growth of Janibacter sp. in MS medium supplemented with nonionic surfactant Tween 80 (40 mg/L) containing 20 and 300 mg/L of PCP.(b) Effect of nonionic surfactant Tween 80 on the PCP removal efficiency by Janibacter sp. Error bars represent the standard deviation. actinobacterium belonging to the genus Janibacter sp., with substrates metabolism. In fact, the NADH provided by respect to morphological and biochemical tests and 16S glucose metabolism may increase the biomass and thus rRNA gene sequence. Despite their known high potential in increase the total activity for PCP metabolizing [46]. recalcitrant compounds biodegradation [29, 33], bacteria of In the present study, PCP removal is affected by pH the genus Janibacter, described in this study, are reported variation. As it was reported by Premalatha and Rajakumar for the first time for their ability to degrade PCP. In [43], Wolski et al. [47], Barbeau et al. [48], and Edgehill biodegradation process, glucose is commonly used as an [22]forArthrobacter and different Pseudomonas species, the additional source of carbon and energy and is the most neutral pH was found to be optimal for PCP degradation. metabolizable sugar which supported a maximum growth However, for other bacterial species, such as Sphingomonas [44]. In this context, our results are in agreement with those chlorophenolica, PCP degradation was more important at pH of Singh et al. [45]andSinghetal.[15]whoreportedthe 9.2 [49]. enhancement of bacterial growth and PCP-degradation Temperature is another important environmental factor process using MSM supplemented with 1% of glucose [43]. that can influence the rate of pollutants degradation48 [ ]. This effect can be explained by the connection of the two The optimal temperature for the PCP removal was recorded BioMed Research International 7

∘ ∘ at 30 C, but lower temperatures (25 C) allowed significant 5. Conclusion removal than the upper values. These results were in accordance with those of Wittmann et al. [9]andCrawford In this study, a novel efficient PCP-degrading actinobac- and Mohn [50]. Overall, deviation in pH and tempera- terium (Janibacter sp.) was isolated from saline soil of arid ture from the optimum results in alteration of microbial land and investigated for its physiological characteristics. growth and metabolism, as well as the pollutants properties Janibacter was able to remove high concentration of PCP and [51, 52]. to tolerate fluctuation of NaCl. This removal potential was The effect of different concentrations of PCP on growth of moreover accelerated by the addition of Tween 80. This study thestrainprovedthatthePCPremovalwasmoreefficientat suggested that strain Janibacter sp. could be widely used for low concentrations (20 mg/L). This result was coherent with PCP bioremediation of polluted arid/extreme environments. data of Webb et al. [53] reporting that all strains tested were able to degrade up to 90% of the PCP,when the concentration Conflict of Interests was10mg/L.Moreover,asitwasrevealedbyKarnetal. [16], the ability of PCP removal of Janibacter sp. decreases The authors declare that there is no conflict of interests when PCP concentration was increased. Furthermore, we regarding the publication of this paper. found that the removal ability by Janibacter sp. has reached 40% after 144 h of incubation, when PCP concentration of 300 mg/L was used. These results are in accordance with Acknowledgments those of Chandra et al. [54]forBacillus cereus strain and This work was financially supported by the NATO Project may suggest that these bacteria may tolerate and remove high SFP (ESP.MD.FFP981674) 0073 “Preventive and Remediation concentrations of PCP if we increase the incubation time. On Strategies for Continuous Elimination of Poly-Chlorinated thecontrary,Kaoetal.[55]reportedthatnoPCPremovalwas Phenols from Forest Soils and Groundwater.” It was in part detected with PCP concentrations of 320 mg/L even after 20 further supported by the European Union in the ambit of days of incubation. the Project ULIXES (European Community’s Seventh Frame- As for bioremediation, the strain should possess not only work Programme, KBBE-2010-4 under Grant Agreement no. the high removal efficiency for the target compounds but 266473). also the strong abilities of adapting some conditions such aspH,temperature,andsalinityfluctuations.Inthisstudy, it was shown that Janibacter sp. was able to remove PCP References even with salinity fluctuations (less than 10%). These results [1] A. Vallecillo, P. A. Garcia-Encina, and M. Pena,˜ “Anaerobic were in accordance with those of Gayathri and Vasudevan biodegradability and toxicity of chlorophenols,” Water Science [56] suggesting that the reduction in phenolic components and Technology,vol.40,no.8,pp.161–168,1999. removal efficiency above 10% NaCl may be due to increase [2] S. Fetzner and F. Lingens, “Bacterial dehalogenases: biochem- in salinity. These results indicated that Janibacter sp. strain istry, genetics, and biotechnological applications,” Microbiolog- has an inherent flexibility to adapt to salinity fluctua- ical Reviews,vol.58,no.4,pp.641–685,1994. tions. [3]C.M.Kao,C.T.Chai,J.K.Liu,T.Y.Yeh,K.F.Chen,andS. The use of surfactants has the potential to increase the C. Chen, “Evaluation of natural and enhanced PCP biodegrada- biodegradation rate of hydrophobic organic compounds in tion at a former pesticide manufacturing plant,” Water Research, contaminated environments. Nonionic surfactants are usu- vol.38,no.3,pp.663–672,2004. ally used in the bioavailability studies due to their relatively [4] D.-S. Shen, X.-W. Liu, and H.-J. Feng, “Effect of easily degrad- low toxicity compared to ionic surfactants [57]. The enhanced able substrate on anaerobic degradation of pentachlorophenol biodegradation in the micelles solution can be attributable in an upflow anaerobic sludge blanket (UASB) reactor,” Journal to the increased solubility, dissolution, and bioavailability of Hazardous Materials,vol.119,no.1–3,pp.239–243,2005. of compound to bacteria [58] and the surfactant enhanced [5]K.Sai,K.S.Kang,A.Hirose,R.Hasegawa,J.E.Trosko,andT. substratetransportthroughthemicrobialcellwall[59]. The Inoue, “Inhibition of apoptosis by pentachlorophenol in v-myc- effects of the surfactants on PCP removal have been invari- transfected rat liver epithelial cells: relation to down-regulation ably attributed to the increased solubility and dissolution of of gap junctional intercellular communication,” Cancer Letters, PCP or enhancement of mass transport in the presence of vol. 173, no. 2, pp. 163–174, 2001. surfactants. In this context, at high concentration of PCP [6] EPA, “Final determination and indent to cancel and deny (300 mg/L), Tween 80 increases the removal rate of PCP when applications for registrations of pesticide products contain- the Tween 80 concentration is 40 mg/L. The enhancement of ing pentachlorophenol (including but not limited to its salts PCP removal was slightly detected when the concentration and esters) for non-wood uses,” US Environmental Protection ofPCPis20mg/L.Theseresultscanbeconfirmedwiththe Agency. Federal Register, vol. 52, pp. 2282–2293, 1987. study of Cort et al. [60] when the biodegradation rate of [7] S. D. Copley, “Evolution of a metabolic pathway for degradation PCP was enhanced for the concentration of PCP at 140 and of a toxic xenobiotic: the patchwork approach,” Trends in 220 mg/L but it was inhibited for the concentration of PCP Biochemical Sciences, vol. 25, no. 6, pp. 261–265, 2000. at 100 and 50 mg/L. Consequently, successful integration of [8] D. Errampalli, J. T. Trevors, H. Lee et al., “Bioremediation: a PCP and Tween 80 degradation was achieved by Janibacter perspective,” Journal of Soil Contamination,vol.6,no.3,pp. sp. strain. 207–218, 1997. 8 BioMed Research International

[9] C. Wittmann, A. P. Zeng, and W. D. Deckwer, “Physiological Journal of Systematic Bacteriology,vol.47,no.2,pp.529–534, characterization and cultivation strategies of the pentachloro- 1997. phenol-degrading bacteria Sphingomonas chlorophenolica [25] J.-H. Yoon, K.-C. Lee, S.-S. Kang, Y.-H. Kho, K.-H. Kang, and RA2 and Mycobacterium chlorophenolicum PCP-1,” Journal Y.-H. Park, “Janibacter terrae sp. nov., a bacterium isolated from of Industrial Microbiology and Biotechnology,vol.21,no.6,pp. soil around a wastewater treatment plant,” International Journal 315–321, 1998. of Systematic and Evolutionary Microbiology,vol.50,no.5,pp. [10] I.S.Thakur,R.Sharma,S.Shukla,andA.H.Ahmed,“Enumera- 1821–1827, 2000. tion and enrichment of pentachlorophenol degrading bacterial [26] J.-H. Yoon, H. B. Lee, S.-H. Yeo, and J.-E. Choi, “Janibacter mel- of pulp and paper miller in the chemostat,” in Proceedings of onis sp. nov., isolated from abnormally spoiled oriental melon the AMI Conference,vol.159,pp.25–27,Jaipur,India,November in Korea,” International Journal of Systematic and Evolutionary 2000. Microbiology, vol. 54, no. 6, pp. 1975–1980, 2004. [11] J. H. A. Apajalahti, P. Karpanoja, and M. S. Salkinoja-Salonen, [27]A.Kageyama,Y.Takahashi,M.Yasumoto-Hirose,H.Kasai, “Rhodococcus chlorophenolicus sp. nov., a chlorophenol-mine- Y. Shizuri, and S. Omura, “Janibacter corallicola sp. nov., iso- ralizing actinomycete,” International Journal of Systematic Bac- lated from coral in Palau,” Journal of General and Applied teriology,vol.36,no.2,pp.246–251,1986. Microbiology,vol.53,no.3,pp.185–189,2007. [12] J. F. Gonzalez and W. S. Hu, “Effect of glutamate on the degra- [28] P. Kampfer,¨ O. Terenius, J. M. Lindh, and I. Faye, “Janibacter dation of pentachlorophenol by Flavobacterium sp,” Applied anophelis sp. nov., isolated from the midgut of Anopheles Microbiology and Biotechnology,vol.35,no.1,pp.100–110,1991. arabiensis,” International Journal of Systematic and Evolutionary [13] R. I. Dams, G. I. Paton, and K. Killham, “Rhizoremediation Microbiology,vol.56,no.2,pp.389–392,2006. of pentachlorophenol by Sphingobium chlorophenolicum ATCC [29] I. Sierra, J. L. Valera, M. L. Marina, and F. Laborda, “Study 39723,” Chemosphere, vol. 68, no. 5, pp. 864–870, 2007. of the biodegradation process of polychlorinated biphenyls in [14] C.-F. Yang, C.-M. Lee, and C.-C. Wang, “Isolation and physi- liquid medium and soil by a new isolated aerobic bacterium ological characterization of the pentachlorophenol degrading (Janibacter sp.),” Chemosphere,vol.53,no.6,pp.609–618,2003. bacterium Sphingomonas chlorophenolica,” Chemosphere,vol. [30] S. Iwai, A. Yamazoe, R. Takahashi, F. Kurisu, and O. Yagi, 62,no.5,pp.709–714,2006. “Degradation of monochlorinated dibeno -p-Dioxins by Jani- [15]S.Singh,B.B.Singh,R.Chandra,D.K.Patel,andV.Rai, bacter sp.strainYAisolatedfromriversediment,”Current “Synergistic biodegradation of pentachlorophenol by Bacillus Microbiology,vol.51,no.5,pp.353–358,2005. cereus (DQ002384), Serratia marcescens (AY927692) and Serra- [31] S. Jin, T.Zhu, X. Xu, and Y.Xu, “Biodegradation of dibenzofuran tia marcescens (DQ002385),” World Journal of Microbiology and by Janibacter terrae strain XJ-1,” Current Microbiology,vol.53, Biotechnology,vol.25,no.10,pp.1821–1828,2009. no. 1, pp. 30–36, 2006. [16]S.K.Karn,S.K.Chakrabarty,andM.S.Reddy,“Pen- [32] A. Yamazoe, O. Yagi, and H. Oyaizu, “Degradation of polycyclic tachlorophenol degradation by Pseudomonas stutzeri CL7 in aromatic hydrocarbons by a newly isolated dibenzofuran- the secondary sludge of pulp and paper mill,” Journal of utilizing Janibacter sp. strain YY-1,” Applied Microbiology and Environmental Sciences, vol. 22, no. 10, pp. 1608–1612, 2010. Biotechnology,vol.65,no.2,pp.211–218,2004. [17]A.Sharma,I.S.Thakur,andP.Dureja,“Enrichment,isola- [33] A. Yamazoe, O. Yagi, and H. Oyaizu, “Biotransformation of fluo- tion and characterization of pentachlorophenol degrading bac- rene, diphenyl ether, dibenzo-p-dioxin and carbazole by Jani- terium Acinetobacter sp. ISTPCP-3 from effluent discharge site,” bacter sp,” Biotechnology Letters,vol.26,no.6,pp.479–486, Biodegradation,vol.20,no.5,pp.643–650,2009. 2004. [34]G.-Y.Zhang,J.-Y.Ling,H.-B.Sun,J.Luo,Y.-Y.Fan,andZ.-J. [18] M. Kamekura, “Diversity of extremely halophilic bacteria,” Cui, “Isolation and characterization of a newly isolated poly- Extremophiles,vol.2,no.3,pp.289–295,1998. cyclic aromatic hydrocarbons-degrading Janibacter anophelis [19] R. Margesin and F. Schinner, “Potential of halotolerant and strain JY11,” Journal of Hazardous Materials,vol.172,no.2-3,pp. halophilic microorganisms for biotechnology,” Extremophiles, 580–586, 2009. vol. 5, no. 2, pp. 73–83, 2001. [35] D. A. Edwards, R. G. Luthy, and Z. Liu, “Solubilization of poly- [20] W. D. Grant, R. T. Gemmell, and T. J. McGenity, “Halophiles,” cyclic aromatic hydrocarbons in micellar nonionic surfactant in Extremophiles: Microbial Life in Extreme Environments,K. solutions,” Envirenmental Science and Technology,vol.25,no.1, Horikoshi and W. D. Grant, Eds., pp. 93–132, Wiley-Liss, New pp.127–133,1991. York, NY, USA, 1998. [36] C. Rosch,¨ A. Mergel, and H. Bothe, “Biodiversity of denitrifying [21] C. K. Okoro, R. Brown, A. L. Jones et al., “Diversity of culturable and dinitrogen-fixing bacteria in an acid forest soil,” Applied and actinomycetes in hyper-arid soils of the Atacama Desert, Chile,” Environmental Microbiology,vol.68,no.8,pp.3818–3829,2002. Antonie van Leeuwenhoek,vol.95,no.2,pp.121–133,2009. [37] K. Wilson, “Preparation of genomic DNA from bacteria,” in [22] R. U. Edgehill, “Pentachlorophenol removal from slightly acidic Current Protocols in Molecular Biology,F.M.Ausubel,R.Brent, mineral salts, commercial sand, and clay soil by recovered R. E. Kingston et al., Eds., pp. 241–245, 1987. Arthrobacter strain ATCC 33790,” Applied Microbiology and [38] D. Daffonchio, S. Borin, G. Frova, P. L. Manachini, and C. Biotechnology,vol.41,no.1,pp.142–148,1994. Sorlini, “PCR fingerprinting of whole genomes: the spacers [23]S.K.Karn,S.K.Chakrabarti,andM.S.Reddy,“Degradationof between the 16s and 23S rRNA genes and of intergenic tRNA pentachlorophenol by Kocuria sp. CL2 isolated from secondary gene regions reveal a different intraspecific genomic variability sludge of pulp and paper mill,” Biodegradation,vol.22,no.1,pp. of Bacillus cereus and Bacillus licheniformis,” International Jour- 63–69, 2011. nal of Systematic Bacteriology, vol. 48, no. 1, pp. 107–116, 1998. [24] K. Martin, P.Schumann, F. A. Rainey, B. Schuetze, and I. Groth, [39] S.F.Altschul,W.Gish,W.Miller,E.W.Myers,andD.J.Lipman, “Janibacter limosus gen. nov., sp. nov., a new actinomycete “Basic local alignment search tool,” Journal of Molecular Biology, with meso- diaminopimelic acid in the cell wall,” International vol. 215, no. 3, pp. 403–410, 1990. BioMed Research International 9

[40] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and [56] K. V. Gayathri and N. Vasudevan, “Enrichment of phenol S. Kumar, “MEGA5: molecular evolutionary genetics analysis degrading moderately halophilic bacterial consortium from using maximum likelihood, evolutionary distance, and max- saline environment,” Journal of Bioremediation & Biodegrada- imum parsimony methods,” Molecular Biology and Evolution, tion,vol.2011,article104,2010. vol. 28, no. 10, pp. 2731–2739, 2011. [57]D.H.Yeh,K.D.Pennell,andS.G.Pavlostathis,“Toxicity [41] N. Saitou and M. Nei, “The neighbor-joining method: a new and biodegradability screening of nonionic surfactants using method for reconstructing phylogenetic trees.,” Molecular biol- sediment-derived methanogenic consortia,” Water Science and ogy and evolution,vol.4,no.4,pp.406–425,1987. Technology,vol.38,no.7,pp.55–62,1998. [42] I. S. Thakur, “Structural and functional characterization of a sta- [58] F. Volkering, A. M. Breure, J. G. van Andel, and W. H. Rul- ble, 4-chlorosalicylic-acid-degrading, bacterial community in a kens, “Influence of nonionic surfactants on bioavailability and chemostat,” WorldJournalofMicrobiologyandBiotechnology, biodegradation of polycyclic aromatic hydrocarbons,” Applied vol. 11, no. 6, pp. 643–645, 1995. and Environmental Microbiology,vol.61,no.5,pp.1699–1705, [43] A. Premalatha and G. S. Rajakumar, “Pentachlorophenol degra- 1995. dation by Pseudomonas aeruginosa,” World Journal of Microbi- [59]W.H.Noordman,J.H.J.Wachter,G.J.deBoer,andD.B. ology and Biotechnology,vol.10,no.3,pp.334–337,1994. Janssen, “The enhancement by surfactants of hexadecane degra- [44] M. Tripathi and S. K. Garg, “Studies on selection of efficient bac- dation by Pseudomonas aeruginosa varies with substrate avail- terial strain simultaneously tolerant to hexavalent chromium ability,” Journal of Biotechnology,vol.94,no.2,pp.195–212,2002. and pentachlorophenol isolated from treated tannery effluent,” [60] T.L. Cort, M.-S. Song, and A. R. Bielefeldt, “Nonionic surfactant Research Journal of Microbiology,vol.5,no.8,pp.707–716,2010. effects on pentachlorophenol biodegradation,” Water Research, [45] S. Singh, R. Chandra, D. K. Patel, and V. Rai, “Isolation vol. 36, no. 5, pp. 1253–1261, 2002. and characterization of novel Serratia marcescens (AY927692) for pentachlorophenol degradation from pulp and paper mill waste,” WorldJournalofMicrobiologyandBiotechnology,vol.23, no. 12, pp. 1747–1754, 2007. [46] M. Cai and L. Xun, “Organization and regulation of pentachlorophenol-degrading genes in Sphingobium chlorophenolicum ATCC 39723,” Journal of Bacteriology,vol.184,no.17,pp.4672– 4680, 2002. [47]E.A.Wolski,S.E.Murialdo,andJ.F.Gonzalez,“Effectof pH and inoculum size on pentachlorophenol degradation by Pseudomonas sp,” Water SA,vol.32,no.1,pp.93–98,2006. [48] C. Barbeau, L. Deschenes,ˆ D. Karamanev, Y. Comeau, and R. Samson, “Bioremediation of pentachlorophenol-contaminated soil by bioaugmentation using activated soil,” Applied Microbi- ology and Biotechnology,vol.48,no.6,pp.745–752,1997. [49] C.-F. Yang, C.-M. Lee, and C.-C. Wang, “Degradation of chlorophenols using pentachlorophenol-degrading bacteria Sphingomonas chlorophenolica in a batch reactor,” Current Microbiology,vol.51,no.3,pp.156–160,2005. [50] R. L. Crawford and W. W. Mohn, “Microbiological removal of pentachlorophenol from soil using a Flavobacterium,” Enzyme and Microbial Technology,vol.7,no.12,pp.617–620,1985. [51] J. T. Trevors, “Effect of temperature on the degradation of pentachlorophenol by Pseudomonas species,” Chemosphere,vol. 11, no. 4, pp. 471–475, 1982. [52] M. A. Providenti, H. Lee, and J. T. Trevors, “Selected factors limiting the microbial degradation of recalcitrant compounds,” Journal of Industrial Microbiology,vol.12,no.6,pp.379–395, 1993. [53] M. D. Webb, G. Ewbank, J. Perkins, and A. J. McCarthy, “Metabolism of pentachlorophenol by Saccharomonospora viridis strains isolated from mushroom compost,” Soil Biology and Biochemistry,vol.33,no.14,pp.1903–1914,2001. [54] R. Chandra, A. Ghosh, R. K. Jain, and S. Singh, “Isolation and characterization of two potential pentachlorophenol degrading aerobic bacteria from pulp paper effluent sludge,” The Journal of General and Applied Microbiology,vol.52,no.2,pp.125–130, 2006. [55]C.M.Kao,J.K.Liu,Y.L.Chen,C.T.Chai,andS.C.Chen, “Factors affecting the biodegradation of PCP by Pseudomonas mendocina NSYSU,” Journal of Hazardous Materials,vol.124, no. 1–3, pp. 68–73, 2005. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 909312, 7 pages http://dx.doi.org/10.1155/2014/909312

Review Article Biotechnological Applications Derived from Microorganisms of the Atacama Desert

Armando Azua-Bustos1 and Carlos González-Silva2 1 Blue Marble Space Institute of Science, Seattle, WA 98109, USA 2 Centro de Investigacion´ del Medio Ambiente (CENIMA), Universidad Arturo Prat, 1110939 Iquique, Chile

Correspondence should be addressed to Armando Azua-Bustos; [email protected]

Received 7 April 2014; Revised 29 June 2014; Accepted 7 July 2014; Published 23 July 2014

Academic Editor: Ameur Cherif

Copyright © 2014 A. Azua-Bustos and C. Gonzalez-Silva.´ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The Atacama Desert in Chile is well known for being the driest and oldest desert on Earth. For these same reasons, it isalso considered a good analog model of the planet Mars. Only a few decades ago, it was thought that this was a sterile place, but in the past years fascinating adaptations have been reported in the members of the three domains of life: low water availability, high UV radiation, high salinity, and other environmental stresses. However, the biotechnological applications derived from the basic understanding and characterization of these species, with the notable exception of copper bioleaching, are still in its infancy, thus offering an immense potential for future development.

1. Introduction as the underside of quartz rocks [8], fumaroles at the Andes Mountains [9],theinsideofhaliteevaporites[10], and caves The Atacama Desert, located in northern Chile between of the Coastal Range [11, 12] showed that microbial life found ∘ ∘ latitudes 17 and 27 south, has average annual rains of less novel ways to adapt to the extreme conditions typical of than 2 mm [1]. In comparison, other known deserts in the the Atacama: extremely low water availability, intense solar world, like the Mojave Desert in North America [2]orthe radiation, and high salinity (for a more complete description Sahara Desert in Africa [3], have average annual rains of of Atacama’s microbial species, please see our recent review 116 mm and 100 mm, respectively. These extremely low rain on this subject [7]).However,uptodate,veryfewworkshave rates have determined the Atacama Desert to be classified as gone beyond the descriptive stage of establishing what types a hyperarid desert [4] (a desert with an aridity index of less of microorganisms may be found in specific microenviron- than 0.05, as the evapotranspiration of water from its soils is ments [13], thus explaining the incipient biotechnological much higher than the inputs of rains). The Atacama Desert is applications derived from knowledge that still being gained. also unique as it is believed to be the oldest desert on Earth, The study of the molecular strategies used by microbial beingaridforthelast150millionyearsandhyperaridforthe life in other extreme environments (high temperature, for past 15 million years [5, 6]. example) gave rise to many biotechnological applications that Thus, the Atacama has been an extremely dry desert are now of standard use [14]. In a similar way, the characteri- for a very long time and only forty years ago it was zation of the molecular strategies evolved by microorganisms thought that nothing could live in its seemingly barren of the Atacama to cope with its exceptional abiotic stresses landscapes (Figure 1(a)). However, during the past ten years, (desiccation in particular) should be multiple and unique, culture dependent and independent methods have unveiled and, thus, novel sources of metabolites and genes for the a plethora of microorganisms (Bacteria, Archaea, and biotechnological industry. In this review, the few reported Eukarya) that were able to adapt and evolve in very specific cases of the biotechnological use of Atacama Desert microor- and unexpected habitats of this desert [7]. Habitats as diverse ganismstodatearesummarized. 2 BioMed Research International

(a) (b)

(c) Figure 1: Examples of habitats of the Atacama Desert from where biotechnological applications have been derived or used. (a) The central valley, the hyperarid core of the Atacama Desert. (b) Heap bioleaching at Radomiro Tomic, an open pit copper mine owned by the Chilean Copper Corporation (Codelco). Note the copper rich blue-green solution obtained from the heaps. (c) The Loa River, a typical arsenic rich river of the Atacama Desert. Image credits: Panels A and C: Armando Azua-Bustos. Panel B: Armando Azua Aroz.

2. Applications Derived from Members of In Chile, the first mine that introduced bioleaching was the Bacteria Domain Sociedad Minera Pudahuel (a copper mine not located in the Atacama) in the 1980s. Today, this process is extensively Copper Bioleaching. Copper bioleaching or “biomining” used in the Chilean copper mining industry [18, 19], reaching allowed the usage of insoluble copper sulphides and oxides over 1.6 million tons of copper per year [19]. It has been through hydrometallurgy, as opposed to the traditional estimated that Chile’s copper actual reserves would increase technology of pyrometallurgy. Compared to pyrometallurgy, up to 50% if all copper sulphides could be economically bioleaching has the advantage of being a simpler process, treated by bioleaching [19]. requiring less energy and equipments (Figure 1(b)). In addi- Acidithiobacillus ferrooxidans hasbeenidentifiedindif- tion, bioleaching does not produce sulfur dioxide emissions, ferent places of the Atacama Desert [20, 21]. Some of these an important factor for the Chilean mining towns which species have been found in sulfidic mine tailings dumps in were usually built alongside the extracting operations in the marine shore at Chanaral˜ Bay [21], located at the Coastal Chile(mostofwhicharelocatedintheAtacamaDesert). Range of the Atacama. This is of particular interest as these Bioleaching also offered a better treatment of low grade species were found to be halotolerant iron oxidizers, active (again the usual case in Atacama copper ores) or waste at NaCl concentrations up to 1 M in enrichment cultures. oresandinmanycasesitistheonlywaytotreatthem. High concentrations of chloride ions inhibit the growth of the Low-gradeores(0.6%andless)areabundantinChile, acidophilic microorganisms traditionally used in biomining but their processing by pyrometallurgy in most cases is [22]. Thus, the finding of halotolerant bioleaching species not economical. Through bioleaching, copper was able to would allow the use of seawater for biomining operations in be extracted from ore minerals like chalcopyrite (CuFeS2), the future, a very important advancement in a region, where with the crucial contribution of chemolithotrophic microbial water availability has always been extremely low. species extremely tolerant to low pH, which use the reduced Bioleaching strains found in the Atacama Desert have sulphur as an energy source. The most known of these been recently patented, as is the case of Acidithiobacillus fer- microorganisms is Acidithiobacillus ferrooxidans [15], but rooxidans strain Wenelen DSM 16786 [23]andAcidithiobacil- other species, like Leptospirillum ferrooxidans, Sulfobacillus lus thiooxidans strain Licanantay DSM 17318 [24](USPatent acidophilus, and Acidimicrobium ferrooxidans, are thought to numbers 7,601,530 and 7,700,343). Both strains showed also participate in the bioleaching process [16, 17]. improved oxidizing activity when compared to standard BioMed Research International 3 strains isolated elsewhere, like Acidithiobacillus ferrooxidans (Staphylococcus aureus, Listeria monocytogenes, and Bacillus ATCC 23270 and Acidithiobacillus thiooxidans ATCC 8085. subtilis), but weak activity against the tested Gram-negative Strain Wenelen, an iron and sulfur oxidizing microorganism, bacteria (E. coli and Vibrio parahaemolyticus). was particularly efficient in oxidizing chalcopyrite, while In a parallel report, they also found that strain C34 strain Licanantay, a strict sulfur oxidizer, showed activity synthetized four new antibiotics of the ansamycin-type in both primary and secondary sulfured minerals, such polyketides with antibacterial activity against both Staphy- as chalcopyrite, covellite, bornite, chalcocite, enargite, and lococcus aureus ATCC 25923 and Escherichia coli ATCC tennantite [24–26]. 25922 [35]. In particular, chaxamycin D4 showed a selective Recently, the comparative genomic analysis and me- antibacterial activity against S. aureus ATCC 25923. Another tabolomic profiles of these two strains were obtained, which of these strains, Streptomyces sp.C38,synthetizedthreenew turned helpful for determining basic aspects of its regulatory macrolactone antibiotics (atacamycins A–C) which exhibited pathways and functional networks, biofilm formation, energy moderate inhibitory activity against the enzyme phospho- control, and detoxification responses [27, 28]. diesterase (PDE-4B2) [36]. Inhibitors of PDE (the most As for Archaea, although some species have been famous of this group being Viagra) can prolong or enhance reported in acid mine drainage in the Atacama [21], there are theeffectsofphysiologicalresponsesmediatedbycAMP yet no reports of strains specifically isolated for industrial use. and cGMP by inhibition of their degradation by PDE and Biomedicine. Soils of the Atacama shelter a number are considered potential therapeutics for pulmonary arterial of bacterial species with promising characteristics for the hypertension, coronary heart disease, dementia, depression, biomedical industry. One of the first descriptions of microor- and schizophrenia [37]. In the case of atacamycin A, it also ganisms that are known to produce such biomolecules was showed anti proliferative activity against cell lines of colon published in 1966 by Cameron et al. [29]. Commissioned by cancer (CXF DiFi), breast cancer (MAXF 401NL), uterus NASA, this group approached the Atacama as a way to obtain cancer (UXF 1138L), and colon RKO cells [36]. basic information on terrestrial desert environments and its Similar positive results were obtained by Leiros´ et al., 2014 microbiota in order to develop and test the instruments to be [38]inwhichsevenmoleculessynthetizedbyStreptomyces taken to Mars ten years later by the Viking Mission. Among sp. Lt 005, Atacama Streptomyces C1, and Streptomyces sp. others, they were among the first to report the presence CBS 198.65 were tested against hydrogen peroxide stress in of Streptomyces species, Bacillus subtilis aterrimus, Bacillus primarycorticalneuronsaspotentiallynewdrugsforthe brevis, Bacillus cereus, and Micrococcus caseolyticus;however, avoidance of neurodegenerative disorders such as Parkin- no details of biomolecules produced by these species were son’s and Alzheimer’s diseases. The reported compounds later reported. inhibited neuronal cytotoxicity and reduced reactive oxygen Almost forty years passed until a groundbreaking report species (ROS) release after 12 h of treatment. Among these by McKay’s group in 2003 [30] showed that when the experi- compounds, the quinone anhydroexfoliamycin and the red ments performed by the Viking landers on the surface of Mars pyrrole-type pigment undecylprodigiosin showed the best were repeated with soils of the Yungay region of the Atacama protection against oxidative stress with mitochondrial func- Desert, the same results were obtained essentially. This leads to the recognition of the Atacama Desert as one of the driest tion improvement, ROS production inhibition, and increase places on Earth, causing then a surge of reports focusing on of antioxidant enzymes like glutathione and catalase. In addi- the characteristics of various microenvironmental conditions tion, both compounds showed a modest caspase-3 activity in the Atacama and its related microbiology [7]. induced by the apoptotic enhancer staurosporine. Later on, the interest in the potential biomedical use of In a different work, another group of secondary metabo- these recently reported species started, focusing on species lites, called abenquines, were found to be synthetized by of the Actinobacteria, as these were previously known as Streptomyces sp. Strain DB634, isolated from the soils of synthesizers of useful molecules [31]. Among this latter class, the Altiplano of the Atacama [39]. These abenquines (A–D) species like Amycolatopsis, Lechevalieria, and Streptomyces showed modest inhibitory activity against Bacillus subtilis, have been reported at various arid and hyperarid sites of the dermatophytic fungi, phosphodiesterase type 4b, and antifi- Atacama [32]. broblast proliferation (NIH-3T3). Members of the Streptomycetes are a well-known source An interesting case to discuss in this section of a commer- of antibiotics [33], and Lechevalieria species are known to cially successful, but controversial, example of a compound have nonribosomal peptide synthase (NRPS) gene clusters produced from an Actinobacteria isolated from another that synthesize antitumoral compounds [31]. Accordingly, well-known Chilean environment is that of rapamycin (also of the species found by Okoro’s group [32], all of the known as sirolimus), isolated by Brazilian researchers from Amycolatopsis and Lechevalieria and most of the Streptomyces astrainofStreptomyces hygroscopicus endemic of Eastern isolates tested positive for the presence of NRPS genes. This Island, or Rapa Nui [40]. Rapamycin was originally used same group determined later the metabolic profile of one as an antibiotic, but later on it was discovered to show of these Streptomyces strains (strain C34), identifying three potent immunosuppressive and antiproliferative properties new compounds from the macrolactone polyketides class [41, 42]andevenclaimedtoextendlifespan[43]. Sadly, [34] and other compounds like deferoxamine E, hygromycin nothing of this development benefited the Chilean economy, 󸀠󸀠 A, and 5 -dihydrohygromycin. These compounds showed as agreements like the United Nations Rio Declaration on a strong activity against the Gram-positive bacteria tested Environment and Development were yet to be established. 4 BioMed Research International

Arsenic Bioremediation. Conventional arsenic removal in additives, and colorants in cosmetics and foods. Interest in drinking water such as reverse osmosis and nanofiltration are dietary carotenoids has increased in the past years due to their effective and able to remove up to 95% of the initial arsenic antioxidant and anti-inflammatory potential [60, 61], as they concentrations, but the operating costs of these plants are are very efficient quenchers of singlet oxygen and scavengers high [44]. In addition, the oxidation of As (III) to As (V) is a of other reactive oxygen species [62]. Carotenoids are also prerequisite for all conventional treatment processes, and as important precursors of retinol (vitamin A) [62, 63]. this is an extremely slow reaction toxic and costly oxidants Among other sources, species of the halophilic biflag- such as chlorine, hydrogen peroxide, or ozone must be used ellate unicellular green alga Dunaliella (Chlorophyta), like as catalysts [44, 45]. Thus, an attractive alternative solution Dunaliella salina, are industrially cultivated as a natural for arsenic removal is bioremediation, as a wide variety of source of beta-carotene around the world, including Chile bacteria can use it as an electron donor for autotrophic [64]. Under conditions of abiotic stress (high salinity, high growth or as an electron acceptor for anaerobic respiration temperature, high light intensity, and nitrogen limitation) up [46–48]. to 12% of the algal dry weight is 𝛽-carotene [65, 66]which In the case of the Atacama Desert, the first steps leading to accumulates in oil globules in the interthylakoid spaces of the biosequestration of arsenic by endemic microorganisms their chloroplast [65]. In addition, as a defense mechanism are now being taken. This toxic metalloid is naturally found in against hypersalinity, D. salina synthetizes high amounts of rivers of the Atacama Desert (Figure 1(b))asarsenateAs(V) thecompatiblesoluteglycerol,anothermoleculeofeconomic and the most toxic species arsenite As (III) [49–51]. Among value [67]. other negative biological effects, arsenate, being a chemical There are several species of the genus Dunaliella reported analog of phosphate, inhibits oxidative phosphorylation and in the Atacama Desert, mainly in hypersaline lagoons [68, 69] arsenite binds to sulfhydryl groups of proteins [52]. It is and even growing aerophytically on cave walls [11]. In the precisely in the sediments of one of these rivers, (Camarones case of D. Salina Isolate Conc-007, isolated from the Salar de rivernearthecoastalcityofArica)witharsenicconcentra- −1 −1 Atacama, it was found to be capable of synthesizing 100 pg tion, in water (1100 𝜇gL ) and sediments (550 𝜇gL )that of beta-carotene per cell, two to four times higher than 49 isolates were identified and distributed between the 𝛼- other species used in commercial beta-carotene production Proteobacteria (5 isolates), 𝛽-Proteobacteria (13 isolates), and [69, 70]. In turn, D. salina SA32007, also isolated from the 𝛾-Proteobacteria (26 isolates) [44, 53]. Most of these species Salar de Atacama, synthesized triglycerides-enriched lipids belonged to the genera Alcaligenes, Burkholderia, Coma- under nitrogen deficiency conditions, a potentially relevant monas, Enterobacter, Erwinia, Moraxella, Pantoea, Serratia, result for biodiesel production [71]. Important differences Sphingomonas,andPseudomonas [53], of which Alcaligenes, in the carotenogenic capacity of the D. salina strains have Burkholderia, Sphingomonas, Pantoea, Erwinia,andSerratia been shown to be dependent on the high genetic diversity were not previously reported in literature as arsenic tolerant. of member of this species [69]. This is highly relevant for Fittingly, eleven of the arsenic-tolerant isolates had the gene the case of the Chilean species, as it seems that they may ars that codes for the critical enzyme involved in this reaction, be better producers of 𝛽-carotene and other biomolecules in arsenate reductase [53]. In a later work from this group, comparison to other species of the world. it was found that one of the species isolated, Pseudomonas An additional factor to consider in this case is that arsenicoxydans strain VC-1, was able to tolerate up to 5 mM Dunaliella production facilities elsewhere (Australia, China, of As (III), being also capable of oxidizing at high rates the andIndia)arelocatedinareaswheresolarirradianceis totality of the arsenite present in the medium, with lactate as a maximal, climate is warm, and hypersaline water is available carbon source [54]. Thus, the characterization of these species [57], which are precisely the characteristics of most areas of in experimental bioreactors will certainly offer interesting the Atacama; thus, growth facilities may well be developed options for future water and soil bioremediation [55]. in this desert using endemic strains. In addition, adaptive laboratory evolution [72] and metabolic engineering may be applied to the Atacama species in the future, as these methods 3. Applications Derived from Microbial have recently been investigated and accomplished [72, 73]. Members of the Eukarya Domain Biomedicine. Carotenoids are lipid soluble tetraterpenoid pig- 4. Final Comments ments synthesized as hydrocarbons (carotene, e.g., lycopene, 𝛼-carotene, and 𝛽-carotene) or their oxygenated derivatives The brevity of this review reflects how little has been advanced (xanthophylls, e.g., lutein, 𝛼-cryptoxanthin, zeaxanthin, etc.) to date in the biotechnological use of members of the by microorganisms and plants [56]. In these organisms, microbial world found in the Atacama Desert. This may be they play multiple and critical roles in photosynthesis, understood. Although the Atacama is well known for its by maintaining the structure and function of photosyn- extreme dryness, up to 2003, there was little interest in explor- thetic complexes, contributing to light harvesting, quenching ing and characterizing its potential microbial ecosystems, as it chlorophyll triplet states, scavenging reactive oxygen species, was generally supposed to be sterile. Ten years later, microbial and dissipating excess energy [57, 58]. Up to date, more than life has been found in most if not all of its habitats, from 700 carotenoids have been described [59]. Yellow, orange, high thermal springs on the Andes Mountains to caves of the and red carotenoids are used as pharmaceuticals, animal feed Coastal Range, thus building a yet ongoing descriptive stage BioMed Research International 5 of extant microbial ecosystems. Therefore, it is not surprising References that the technological stage of research is just beginning. [1] C. P. McKay, E. I. Friedmann, B. Gomez-Silva,´ L. Caceres-´ As previously mentioned, the Atacama Desert is unique Villanueva, D. T. Andersen, and R. Landheim, “Temperature asithasbeenthemostaridplaceonEarthforaverylong and moisture conditions for life in the extreme arid region of time, imposing the same selection pressure over the life forms the atacama desert: four years of observations including the El that arrived and then coevolved with it. Fittingly, all reports Nino˜ of 1997-1998,” Astrobiology,vol.3,no.2,pp.393–406,2003. to date have shown that these species are unique in the way [2]J.F.Reynolds,P.R.Kemp,K.Ogle,andR.J.Fernandez,´ the capture, store and use water, tolerate solar radiation, high “Modifying the ’pulse-reserve’ paradigm for deserts of North saline conditions and low soil nutrients [7, 74]. Thus, with the America: precipitation pulses, soil water, and plant responses,” exception of copper bioleaching, now a multimillion dollar Oecologia,vol.141,no.2,pp.194–210,2004. industry, we believe there is an immense biotechnological [3] C.J.Tucker,H.E.Dregne,andW.W.Newcomb,“Expansionand potential waiting to be discovered and developed in relation contraction of the Sahara desert from 1980 to 1990,” Science,vol. to the tolerance to the aforementioned abiotic stresses. 253,no.5017,pp.299–300,1991. Most groups now are still reporting various degrees of [4] United Nations Environment Programme (UNEP), World Atlas tolerance to abiotic stresses in the frame of basic research of Desertification, 1992. (extreme environments and astrobiology in particular), and [5]A.J.Hartley,G.Chong,J.Houston,andA.E.Mather,“150 we foresee that during the next years the detailed understand- million years of climatic stability: evidence from the Atacama ing of the physiological and molecular mechanisms involved Desert, northern Chile,” Journal of the Geological Society,vol. 162, no. 3, pp. 421–424, 2005. in the many abiotic stress tolerances shown by these species should increase to a great extent (see [13], e.g.). “Omics” [6] J. Houston and A. J. Hartley, “The central andean west-slope rainshadow and its potential contribution to the origin of techniques, like genomics, proteomics, and metabolomics, hyper-aridity in the Atacama Desert,” International Journal of and high-throughput technologies will be key in elucidating Climatology, vol. 23, no. 12, pp. 1453–1464, 2003. processes and mechanisms involved in these tolerances and [7] A. Azua-Bustos, C. Urrejola, and R. Vicuna,˜ “Life at the dry then in identifying and characterizing key molecules of edge: microorganisms of the Atacama Desert,” The FEBS Letters, potential use. vol. 586, no. 18, pp. 2939–2945, 2012. In the case of bioleaching, we envision that new bacte- [8] A. Azua-Bustos,´ C. Gonzalez-Silva,´ R. A. Mancilla et al., rial and archaeal strains will appear in the market. Tailor- “Hypolithic cyanobacteria supported mainly by fog in the made combinations of mine-specific strains will probably be coastal range of the Atacama Desert,” Microbial Ecology,vol.61, isolated, characterized, and patented in order to maximize no. 3, pp. 568–581, 2011. the dissolution of copper from the particular complexity [9]E.K.Costello,S.R.P.Halloy,S.C.Reed,P.Sowell,andS.K. of minerals characteristic of each place. In addition, other Schmidt, “Fumarole-supported islands of biodiversity within a propertiesoftheminemaybetakenintoaccountinthe hyperarid, high-elevation landscape on socompa volcano, puna determination of the characteristics of the strain mixture, like de atacama, andes,” AppliedandEnvironmentalMicrobiology, water quality, soil temperature, and so forth. vol.75,no.3,pp.735–747,2009. [10] J. Wierzchos, C. Ascaso, and C. P.McKay, “Endolithic cyanobac- In the case of biomolecules of interest for the biomedical teria in halite rocks from the hyperarid core of the Atacama industry, there are already a handful of groups charac- Desert,” Astrobiology,vol.6,no.3,pp.415–422,2006. terizing potentially interesting biomolecules from bacterial [11] A. Azua-Bustos,´ C. Gonzalez-Silva,L.Salas,R.E.Palma,andR.´ strains isolated from the hyperarid areas and hypersaline Vicuna,˜ “A novel subaerial Dunaliella species growing on cave lagoons of the Atacama Desert, biomolecules which have spiderwebs in the Atacama Desert,” Extremophiles,vol.14,no. just been identified, and their activities preliminary tested. 5,pp.443–452,2010. These isolates are few and are representatives of a very small [12] A. Azua-Bustos,´ C. Gonzalez-Silva,´ R. A. Mancilla et al., fraction of the habitats of the Atacama, so novel strains “Ancient photosynthetic eukaryote biofilms in an Atacama and metabolites will certainly appear in the near future. Desert coastal cave,” Microbial Ecology,vol.58,no.3,pp.485– Microorganisms of the dry core of the Atacama will be of 496, 2009. particular interest, as we expect that these species, being [13] A. Azua-Bustos, J. Zu´niga,˜ C. Arenas-Fajardo, M. Orellana, subjected to the most extreme conditions, should produce L. Salas, and R. Vicuna,˜ “Gloeocapsopsis AAB1, an extremely a number of biomolecules involved in the competition for desiccation-tolerant cyanobacterium isolated from the Ata- scarce resources. cama Desert,” Extremophiles,vol.18,no.1,pp.61–74,2014. With the increasing pressure of finding new drugs able [14] P. L. Bergquist, H. W. Morgan, and D. Saul, “Selected enzymes to handle antibiotic-resistant pathogens [75], extreme envi- from extreme thermophiles with applications in biotechnology,” Current Biotechnology,vol.3,no.1,pp.45–59,2014. ronments are now being investigated in detail [76], and the Atacama Desert, given its unique peculiarities, may be a [15] D. P. Kelly and A. P. Wood, “Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus prime place to explore. gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov.,” International Journal of Systematic and Evolutionary Conflict of Interests Microbiology,vol.50,no.2,pp.511–516,2000. [16] P. Norris, “Acidophile diversity in mineral sulfide oxidation,” The authors declare that there is no conflict of interests in Biomining,D.RawlingsandB.Johnson,Eds.,pp.199–216, regarding the publication of this paper. Springer, Berlin, Germany, 2007. 6 BioMed Research International

[17] A. Schippers, “Microorganisms involved in bioleaching and [31] H. Onaka, “Biosynthesis of heterocyclic antibiotics in actino- nucleic acid-based molecular methods for their identification mycetes and an approach to synthesize the natural compounds,” and quantification,” in MicrobialProcessingofMetalSulfides,E. Actinomycetologica,vol.20,no.2,pp.62–71,2006. Donati and W. Sand, Eds., pp. 3–33, Springer, Dordrecht, The [32] C. K. Okoro, R. Brown, A. L. Jones et al., “Diversity of culturable Netherlands, 2007. actinomycetes in hyper-arid soils of the Atacama Desert, Chile,” [18] E. M. Domic-Mihovilovic, “A review of the development and Antonie van Leeuwenhoek, International Journal of General and current status of copper bioleaching operations in Chile: 25 Molecular Microbiology,vol.95,no.2,pp.121–133,2009. years of successful commercial implementation,” in Biomining, [33] R. E. Procopio,I.R.Silva,M.K.Martins,J.L.Azevedo,and´ D.E.RawlingsandB.D.Johnson,Eds.,pp.81–95,Springer, J. M. Araujo,´ “Antibiotics produced by Streptomyces,” Brazilian Berlin, Germany, 2007. Journal of Infectious Diseases,vol.6,no.5,pp.466–471,2012. [19] J. C. Gentina and F. Acevedo, “Application of bioleaching to [34] M. E. Rateb, W. E. Houssen, W. T. A. Harrison et al., “Diverse copper mining in Chile,” Electronic Journal of Biotechnology,vol. metabolic profiles of a Streptomyces strain isolated from a hyper- 16, no. 3, pp. 1–16, 2013. arid environment,” JournalofNaturalProducts,vol.74,no.9,pp. [20] K. P. Drees, J. W. Neilson, J. L. Betancourt et al., “Bacterial 1965–1971, 2011. community structure in the hyperarid core of the Atacama [35] M. E. Rateb, W.E. Houssen, M. Arnold et al., “Chaxamycins A— Desert, Chile,” Applied and Environmental Microbiology,vol.72, D, bioactive ansamycins from a hyper-arid desert Streptomyces no. 12, pp. 7902–7908, 2006. sp.,” Journal of Natural Products,vol.74,no.6,pp.1491–1499, [21] H. Korehi, M. Blothe,M.A.Sitnikova,B.Dold,andA.¨ 2011. Schippers, “Metal mobilization by iron- and sulfur-oxidizing [36] J. Nachtigall, A. Kulik, S. Helaly et al., “Atacamycins A-C, 22- bacteria in a multiple extreme mine tailings in the Atacama membered antitumor macrolactones produced by Streptomyces Desert, Chile,” Environmental Science & Technology,vol.47,no. sp. C38,” JournalofAntibiotics,vol.64,no.12,pp.775–780,2011. 5, pp. 2189–2196, 2013. [37] Y. H. Jeon, Y. Heo, C. M. Kim et al., “Phosphodiesterase: [22] C. M. Zammit, S. Mangold, V. Rao Jonna et al., “Bioleaching in overview of protein structures, potential therapeutic applica- brackish waters-effect of chloride ions on the acidophile pop- tions and recent progress in drug development,” Cellular and ulation and proteomes of model species,” Applied Microbiology Molecular Life Sciences,vol.62,no.11,pp.1198–1220,2005. and Biotechnology,vol.93,no.1,pp.319–329,2012. [38] M. Leiros,E.Alonso,J.A.Sanchezetal.,“Mitigationof´ [23] T. Sugio, A. Miura, P. A. Parada Valdecantos, and R. Badilla ROS insults by Streptomyces secondary metabolites in primary Ohlbaum, “Bacteria strain Wenelen DSM 16786, use of said cortical neurons,” ACS Chemical Neuroscience,vol.15,no.1,pp. bacteria for leaching of ores or concentrates containing metallic 71–80, 2014. sulfide mineral species and leaching processes based on the use [39] D. Schulz, P. Beese, B. Ohlendorf et al., “Abenquines A-D: of said bacteria or other publications mixtures that contain said Aminoquinone derivatives produced by Streptomyces sp. strain bacteria,” United States Patent US 7,601,530 B2, 2009. DB634,” Journal of Antibiotics,vol.64,no.12,pp.763–768,2011. [24] A. Ohata, M. Manaba, and P. A. Parada Valdecantos, “Sulfur- [40] C. Vezina,A.Kudelski,andS.N.Sehgal,“Rapamycin(AY´ oxidizing bacteria and their use in bioleaching processes for 22,989), a new antifungal antibiotic. I. Taxonomy of the produc- sulfured copper minerals,”United States Patent no. US 7,700,343 ing streptomycete and isolation of the active principle,” Journal B2, 2009. of Antibiotics, vol. 28, no. 10, pp. 721–726, 1975. [25] A. Ohata, M. Manabe, and P. A. Parada, “Sulfur-oxidizing [41] B. K. Law, “Rapamycin: an anti-cancer immunosuppressant?” bacteria and their use in bioleaching processes for sulfured Critical Reviews in Oncology/Hematology,vol.56,no.1,pp.47– copper minerals,” United States Patent No. US 7,700,343, 2010. 60, 2005. [26] T. Sugio, A. Miura, P. A. Parada, and R. Badilla, “Bacteria [42] F. Neff, D. Flores-Dominguez, D. P. Ryan et al., “Rapamycin strain Wenelen DSM 16786, use of said bacteria for leaching of extends murine lifespan but has limited effects on aging,” ores or concentrates containing metallic sulfide mineral species Journal of Clinical Investigation,vol.123,no.8,pp.3272–3291, and leaching processes based on the use of said bacteria or 2013. mixtures that contain said bacteria,” United States Patent No. [43] M. V. Blagosklonny, “Rapamycin extends life- and health span US 7,601,530, 2009. because it slows aging,” Aging,vol.5,no.8,pp.592–598,2013. [27] G. Levican,´ J. A. Ugalde, N. Ehrenfeld, A. Maass, and P. Parada, [44]V.L.Campos,G.Escalante,J.Yanez,˜ C. A. Zaror, and M. “Comparative genomic analysis of carbon and nitrogen assim- A. Mondaca, “Isolation of arsenite-oxidizing bacteria from a ilation mechanisms in three indigenous bioleaching bacteria: natural biofilm associated to volcanic rocks of Atacama Desert, predictions and validations,” BMC Genomics,vol.9,article581, Chile,” Journal of Basic Microbiology, vol. 49, no. 1, pp. S93–S97, 2008. 2009. [28] P.Mart´ınez, S. Galvez,´ N. Ohtsuka et al., “Metabolomic study of [45] M. Kim, J. Nriagu, and S. Haack, “Carbonate ions and arsenic Chilean biomining bacteria Acidithiobacillus ferrooxidans strain dissolution by groundwater,” Environmental Science and Tech- Wenelen and Acidithiobacillus thiooxidans strain Licanantay,” nology,vol.34,no.15,pp.3094–3100,2000. Metabolomics,vol.9,no.1,pp.247–257,2013. [46] K. Hrynkiewicz and C. Baum, “Application of microorganisms [29] R. E. Cameron, D. R. Gensel, and G. B. Blank, “Soil studies— in bioremediation of environment from heavy metals,” in desert microflora XII, abundance of microflora in soil samples Environmental Deterioration and Human Health,A.Malik, from the Chile Atacama Desert,” in Space Programs Summary, E.Grohmann,andR.Akhtar,Eds.,pp.215–227,Springer, vol. 4, pp. 37–38, Jet Propulsion Laboratory, 1966. Amsterdam, The Netherlands, 2014. [30] R. Navarro-Gonzalez,F.A.Rainey,P.Molinaetal.,“Mars-like´ [47] S. Yamamura and S. Amachi, “Microbiology of inorganic soils in the Atacama Desert, Chile, and the dry limit of microbial arsenic: from metabolism to bioremediation,” Journal of Bio- life,” Science,vol.302,no.5647,pp.1018–1021,2003. science and Bioengineering,vol.118,no.1,pp.1–9,2014. BioMed Research International 7

[48] J. H. Huang, “Impact of microorganisms on arsenic biogeo- [66] A. Ben-Amotz, “Dunaliella 𝛽-carotene: from science to com- chemistry: a review,” Water, Air, & Soil Pollution,vol.225,no. merce,” in Enigmatic Microorganisms and Life in Extreme 2, article 1848, 2014. Environments, J. Seckbach, Ed., pp. 399–410, Kluwer Academic [49] P. L. Smedley and D. G. Kinniburgh, “A review of the source, Publishers, Dordrecht, The Netherlands, 1999. behaviour and distribution of arsenic in natural waters,” Applied [67] A. Ben-Amotz and M. Avron, “The role of glycerol in the Geochemistry,vol.17,no.5,pp.517–568,2002. osmotic regulation of the halophilic alga Dunaliella parva,” [50] D. K. Nordstrom, “Worldwide occurrences of arsenic in ground Plant Physiology,vol.51,no.5,pp.875–878,1973. water,” Science,vol.296,no.5576,pp.2143–2145,2002. [68] P. Araneda, C. Jimenez, and B. Gomez-Silva,´ “Microalgae from [51] D. D. Caceres, P. Pino, N. Montesinos, E. Atalah, H. Amigo, Northern Chile. III. Growth and beta carotene content of three and D. Loomis, “Exposure to inorganic arsenic in drinking isolates of Dunaliella salina from the Atacama Desert,” Revista water and total urinary arsenic concentration in a Chilean de Biolog´ıa Marina y Oceanograf´ıa,vol.27,no.2,pp.157–162, population,” Environmental Research, vol. 98, no. 2, pp. 151–159, 1992. 2005. [69] P. I. Gomez´ and M. A. Gonzalez,´ “Genetic variation among [52]S.Shen,X.F.Li,W.R.Cullen,M.Weinfeld,andX.C.Le, seven strains of Dunaliella salina (Chlorophyta) with industrial “Arsenic binding to proteins,” Chemical Reviews, vol. 113, no. 10, potential,basedonRAPDbandingpatternsandonnuclearITS pp. 7769–7790, 2013. rDNA sequences,” Aquaculture,vol.233,no.1–4,pp.149–162, [53] G. Escalante, V. L. Campos, C. Valenzuela, J. Yanez,˜ C. Zaror, 2004. andM.A.Mondaca,“Arsenicresistantbacteriaisolatedfrom [70]A.S.Cifuentes,M.Gonzalez,O.Parra,M.Zu,andM.Zuniga,˜ arsenic contaminated river in the Atacama Desert (Chile),” “Culture of strains of Dunaliella salina (Teodoresco 1905) in Bulletin of Environmental Contamination and Toxicology,vol. different media under laboratory conditions,” Revista Chilena 83,no.5,pp.657–661,2009. de Historia Natural,vol.69,no.1,pp.105–112,1996. [54] V. L. Campos, C. Valenzuela, P. Yarza et al., “Pseudomonas [71] D. Arias-Forero, G. Hayashida, M. Aranda et al., “Protocol arsenicoxydans sp nov., an arsenite-oxidizing strain isolated for maximizing the triglycerides-enriched lipids production from the Atacama desert,” Systematic and Applied Microbiology, from Dunaliella salina SA32007 biomass, isolated from the vol. 33, no. 4, pp. 193–197, 2010. Salar de Atacama (Northern Chile),” Advances in Bioscience and [55] A. S. Butt and A. Rehman, “Isolation of arsenite-oxidizing Biotechnology,vol.4,no.8,pp.830–839,2013. bacteria from industrial effluents and their potential usein [72] W. Fu, O. Guðmundsson, G. Paglia et al., “Enhancement of wastewater treatment,” World Journal of Microbiology and carotenoid biosynthesis in the green microalga Dunaliella salina Biotechnology,vol.27,no.10,pp.2435–2441,2011. with light-emitting diodes and adaptive laboratory evolution,” [56] A.Das,S.H.Yoon,S.H.Lee,J.Y.Kim,D.K.Oh,andS.W.Kim, Applied Microbiology and Biotechnology,vol.97,no.6,pp.2395– “An update on microbial carotenoid production: application of 2403, 2013. recent metabolic engineering tools,” Applied Microbiology and [73]D.Geng,Y.Han,Y.Wangetal.,“Constructionofasystemfor Biotechnology,vol.77,no.3,pp.505–512,2007. the stable expression of foreign genes in Dunaliella salina,” Acta [57] J. A. del Campo, M. Garc´ıa-Gonzalez,´ and M. G. Guerrero, Botanica Sinica,vol.46,no.3,pp.342–346,2004. “Outdoor cultivation of microalgae for carotenoid produc- [74] A. Azua-Bustos, C. Gonzalez-Silva,´ C. Arenas-Fajardo, and R. tion: current state and perspectives,” Applied Microbiology and Vicuna,˜ “Extreme environments as potential drivers of conver- Biotechnology,vol.74,no.6,pp.1163–1174,2007. gent evolution by exaptation: the Atacama Desert Coastal Range [58] B. Demmig-Adams and W. W. Adams III, “Antioxidants in case,” Frontiers in Microbiology,vol.3,article426,2012. photosynthesis and human nutrition,” Science, vol. 298, no. [75] B. Cannon, “Microbiology: resistance fighters,” Nature,vol.509, 5601, pp. 2149–2153, 2002. no. 7498, pp. S6–S8, 2014. [59] G. Britton, S. Liaaen-Jensen, and H. Pfander, Carotenoids [76] A. Tasiemski, S. Jung, C. Boidin-Wichlacz et al., “Characteri- Handbook,Birkhaauser,¨ Basel, Switzerland, 2004. zation and function of the first antibiotic isolated from a vent [60] A. Das, S. Yoon, S. Lee, J. Kim, D. Oh, and S. Kim, “An organism: the extremophile metazoan Alvinella pompejana,” update on microbial carotenoid production: application of PLoS ONE,vol.9,no.4,ArticleIDe95737,2014. recent metabolic engineering tools,” Applied Microbiology and Biotechnology,vol.77,no.3,pp.505–512,2007. [61] M. M. Ciccone, F.Cortese, M. Gesualdo et al., “Dietary intake of carotenoids and their antioxidant and anti-inflammatory effects in cardiovascular care, mediators of inflammation,” Mediators of Inflammation, vol. 2013, Article ID 782137, 11 pages, 2013. [62] J. Fiedor J and K. Burda, “Potential role of carotenoids as antioxidants in human health and disease,” Nutrients,vol.6,no. 2, pp. 466–488, 2014. [63] L. H. Reyes, J. M. Gomez, and K. C. Kao, “Improving carotenoids production in yeast via adaptive laboratory evolution,” Metabolic Engineering,vol.21,no.1,pp.26–33,2014. [64] A. Oren, “A hundred years of Dunaliella research: 1905–2005,” Saline Systems,vol.1,no.2,2005. [65]W.Fu,G.Paglia,M.Magnusd´ ottir´ et al., “Effects of abiotic stressors on lutein production in the green microalga Dunaliella salina,” Microbial Cell Factories,vol.13,no.3,pp.1–9,2014. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 439197, 11 pages http://dx.doi.org/10.1155/2014/439197

Research Article Diversity and Enzymatic Profiling of Halotolerant Micromycetes from Sebkha El Melah, a Saharan Salt Flat in Southern Tunisia

Atef Jaouani,1 Mohamed Neifar,1 Valeria Prigione,2 Amani Ayari,1 Imed Sbissi,1 Sonia Ben Amor,1 Seifeddine Ben Tekaya,1 Giovanna Cristina Varese,2 Ameur Cherif,3 and Maher Gtari1

1 Laboratoire Microorganismes et Biomolecules´ Actives, Faculte´ des Sciences de Tunis, Universite´ Tunis El Manar, Campus Universitaire, 2092 Tunis, Tunisia 2 Dipartimento di Scienze della Vita e Biologia dei Sistemi, Universita` degli Studi di Torino, Viale Mattioli 25, 10125 Torino, Italy 3 Laboratoire Biotechnologie et Valorisation des Bio-Geo´ Ressources, Institut Superieur´ de Biotechnologie de Sidi Thabet, Universite´ La Manouba, 2020 Sidi Thabet, Tunisia

Correspondence should be addressed to Atef Jaouani; [email protected]

Received 2 May 2014; Accepted 28 June 2014; Published 16 July 2014

Academic Editor: Sara Borin

Copyright © 2014 Atef Jaouani et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Twenty-one moderately halotolerant fungi have been isolated from sample ashes collected from Sebkha El Melah, a Saharan salt flat located in southern Tunisia. Based on morphology and sequence inference from the internal transcribed spacer regions, 28S rRNA gene and other specific genes such as 𝛽-tubulin, actin, calmodulin, and glyceraldehyde-3-phosphate dehydrogenase, the isolates were found to be distributed over 15 taxa belonging to 6 genera of Ascomycetes: Cladosporium (𝑛=3), Alternaria (𝑛=4), Aspergillus (𝑛=3), Penicillium (𝑛=5), Ulocladium (𝑛=2), and Engyodontium (𝑛=2). Their tolerance to different concentrations of salt in solid and liquid media was examined. Excepting Cladosporium cladosporioides JA18, all isolates were considered as alkalihalotolerant since they were able to grow in media containing 10% of salt with an initial pH 10. All isolates were resistant to oxidative ∘ stresses and low temperature whereas 5 strains belonging to Alternaria, Ulocladium, and Aspergillus genera were able to grow at 45 C. The screening of fungal strains for sets of enzyme production, namely, cellulase (CMCase), amylase, protease, lipase, and laccase, in presence of 10% NaCl, showed a variety of extracellular hydrolytic and oxidative profiles. Protease was the most abundant enzyme produced whereas laccase producers were members of the genus Cladosporium.

1. Introduction on two promising viewpoints: first, as model for deciphering stress adaptation mechanisms in eukaryotes [9]andsec- Sebkhas are salt flats occurring on arid coastline in North ondary, as novel and largely unexplored materials for the Africa, Arabia, Baja California, and Shark Bay Australia [1]. screening of novel bioactive natural products [10]. Over They are considered among the most poikilotopic environ- the past decade, there is an increased awareness for new ments and characterized by extreme salt concentrations and hydrolytic enzymes useful under nonconventional conditions electromagnetic radiation exposure together with low water [11]. and nutrient availabilities [2]. Regarded as detrimental to Sebkha El Melah, a Saharan salt flat of southern Tunisia, 2 “normal subsistence,” organisms copying such conditions to has an area of approximately 150 km and the level is slightly survive and thrive are designed extremophiles [3]. Beside below the sea. Fluvial basin excavation of Sebkha El Melah halophytes plants and algae, the mostly diverse dwellers of appeared at the beginning of the Wurmian¨ Quaternary sebkhas being unveiled are members of bacterial, archaeal, period [12]. Around 40,000 BP the lagoon was highly desali- and fungal ranks [4–8]. Members of fungi kingdom recovered nated by freshwater arrivals. At the upper Wurm,¨ seawater from extreme environments such as sebkhas’ have shed light withdrew and the basin evolves to a temporary lake or 2 BioMed Research International continental sebkha. More recently, around 8000 years BP, thelagoonevolvedintoanevaporitebasin.Thesebkha sediments are composed of several saliferous layers of rock Sea salt and gypsum (calcium sulfate) and/or polyhalite (sulfate Tunisia of potassium, calcium, and magnesium) [12]. Here we report L3 Mediterranean Mediterranean the isolation of moderately halotolerant fungi from Sebkha El Algeria L1 Melah. Strains have been identified based on morphological L2 and molecular markers and their resistance to salt, thermal, Libya alkaline, and oxidative stresses was assessed. Their ability to produce different hydrolytic and oxidative enzymes under salt stress was also evaluated. Figure 1: Map of Sebkha El Melah (Google Earth). L1, L2, and L3 indicate locations of sampling.

2. Material and Methods genus, the actin gene was amplified using the primers ACT- 󸀠 󸀠 2.1. Sampling Site Description and Fungal Isolation. Three 512F (5 -ATGTGCAAGGCCGGTTTCGC-3 )andACT- locations from the Sebkha El Melah margins (L1: 󸀠 󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 783R (5 -TACGAGTCCTTCTGGCCCAT-3 ) according to 33 23 01.1 N1054 56.8 E; L2: 33 21 42.1 N1055 05.5 E; ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 Bensch et al. [22]; for Alternaria genus, the glyceraldehyde- and L3: 33 23 37.7 N10 53 40.2 E) were chosen for sampling 3-phosphate dehydrogenase gene was amplified using the 󸀠 󸀠 (Figure 1). From each location, a composite sample was primers GPD1 (5 -CAACGGCTTCGGTCGCATTG-3 )and 󸀠 󸀠 prepared aseptically from five subsamples (1–10 cm deep) GPD2 (5 -GCCAAGCAGTTGGTTGTGC-3 ) according to and collected from the arms and center of an X (each arm Berbee et al. [23]; for Penicillium and Aspergillus genera, was 1 m in length) [13]. One cm soil from the ground surface the 𝛽-tubulin gene was amplified using the primers Bt2a 󸀠 󸀠 was firstly removed to avoid contamination during sampling (5 -GGTAACCAAATCGGTGCTGCTTTC-3 )andBt2B procedure. Samples were then transported to the laboratory 󸀠 󸀠 ∘ (5 -ACCCTCAGTGTAGTGACCCTTGGC-3 ) according to in a cool box and stored at 4 Cpriortoprocessing. Glass and Donaldson [24]. Fungi were isolated on potato dextrose agar (PDA) ThePCRproductswerepurifiedwithQIAquickWizard containing 10% of NaCl and 0.05% of chloramphenicol using PCR purification Kit (Promega) according to the manu- the soil plate method where few milligrams of sample were facturer’s instructions, and the sequences were determined directly spread on the agar medium. This method has a slight by cycle sequencing using the Taq Dye Deoxy Termi- edge over the dilution plate method since it allows higher nator Cycle Sequencing kit (Applied Biosystems, HTDS, total number of isolates and limits invasion by species which Tunisia) and fragment separation in an ABI PrismTM 3130 sporulate heavily [14]. DNA sequencer (Applied Biosystems, HTDS, Tunisia). The sequences obtained were compared reference sequences in the NCBI GenBank database using the BLASTN search 2.2. Morphological and Molecular Identification. Isolated option [25]. fungi were identified conventionally according to their macroscopic and microscopic features. After determination 2.3. Effect of pH, Salinity, Temperature, and Oxidative Stress. of their genera [15–17], they were transferred to the media PDA medium was used to study the effect of different stresses recommended of selected genus monographs for species on solid media. For oxidative stresses, H2O2 or paraquat was identification. filter sterilized and added separately to melted PDA medium DNA extraction was achieved as described by Liu et previously autoclaved. Paraquat is a redox-cycling agent al. [18]; the amplification of the internal transcribed spacer widely used to generate reactive oxygen species and induce regions (nuclear-encoded 18S rRNA-ITS1-5.8S rRNA-ITS2- oxidative stress in bacteria [26]andfungi[27]. For pH stress, 28S rRNA) was performed using the couple of universal PDA medium was buffered with 100 mM Glycine-NaOH to 󸀠 󸀠 primers ITS1 (5 -TCC GTA GGT GAA CCT GCG G-3 )and pH 10 before autoclaving. Salt stress in solid media was 󸀠 󸀠 ITS4 (5 -TCC TCC GCT TAT TGA TAT GC-3 )[19]and studied in PDA medium containing different concentrations the thermal cycler conditions according to Luo and Mitchell of salts. The inoculated plates with 3 mm cylindrical mycelial ∘ [20]. PCR was carried out in 25 𝜇L volumes containing 2.5 𝜇L plugs were then incubated at 30 Cforoxidative,salt,and ∘ ∘ of 1X PCR reaction buffer (100 mM Tris-HCl, 500 mM KCl, pH stresses and at 4 Cand45C for thermal stresses, and pH 8.3), 1.5 𝜇L MgCl2,0.2𝜇mol/L (each) primer, 0.2 𝜇mol/L radial growth was measured daily. Results were expressed (each) dNTP, and 2.5 units of Taq polymerase (Dream Taq, as relative growth of fungal strains under different stresses Fermentas) and 1 𝜇L of DNA template. Depending on the as follows: (Colony diameter under stress/colony diameter fungus genus, different gene sequences were amplified. For without stress after 7 days incubation) × 100. the Aspergillus flavus group, the calmodulin gene was ampli- The effect of salinity in liquid medium was carried out in 󸀠 fied using the primers CL1 (5 -GARTWCAAGGAGGCC- Biolog system, a commercially redox based test (Biolog Inc., 󸀠 󸀠 TTCTC-3 )andCL2A(5-TTTTGCATCATGAGTTGGAC- Hayward, CA). Malt extracts (2%) containing 0%, 5%, 10%, 󸀠 3 ) according to Rodrigues et al. [21]; for the Cladosporium 15%, and 20% of salt were inoculated by a suspension of spores BioMed Research International 3 and fragmented mycelium according to the supplier’s instruc- been deposited at the Mycotheca Universitatis Taurinensis tions in 96-well microtiter plates. After 15 days incubation (MUT) in the University of Turin. ∘ at 30 C, the numeric results were extracted using PM Data Analysis 1.3 software. The fungal growth was assimilated to 3.2. Salt Tolerance of Fungal Isolates. Salt tolerance of the the reduction of the redox indicator. The ability of the fungus fungal isolates was assessed on solid and liquid media for togrowinthepresenceofsaltwasexpressedastheratioof NaCl content ranging from 5 to 20%. In solid media, salt kinetic curve surface under stress versus without stress. tolerance was estimated as relative growth represented by the ratio of colony diameter under salt stress to that without 2.4. Extracellular Enzymes Production Profiling. The capacity salt stress. As illustrated in Table 2,alltheisolatedstrains of fungal isolates to produce extracellular enzymes, namely, succeeded to grow in the presence of 10% of salt. While 19 amylase, cellulase, protease, laccase and lipase, was assayed isolates remain able to grow under 15% NaCl, only 7 isolates inthepresenceof10%ofNaCl.Inoculationwasmade tolerated 20% NaCl: Penicillium chrysogenum JA1 and JA22, by transferring 3 mm of cylindrical mycelial plugs on the Cladosporium halotolerans JA8, Cladosporium sphaerosper- corresponding media. Amylase production was assayed on mum JA2, Cladosporium cladosporioides JA18,Aspergillus PDA containing 1% soluble starch. Enzyme production is flavus JA4, and Engyodontium album JA7. shown by the presence of clear halo when iodine was When liquid cultures were used, fungal isolates seemed poured onto the plates. Cellulase production was tested to become more sensitive to salt stress. Indeed, none of the on PDA medium containing 1% of carboxymethylcellulose. strains was able to grow in the presence of 20% NaCl, whereas The presence of activity is reflected by a clear halo on red only 8 strains and 19 strains tolerated 15% and 10% NaCl, background after flooding the plates with 0.2% Congo red respectively (Table 2). for 30 min. Protease production was revealed on skim milk agar by the appearance of a clear zone corresponding to 3.3. Alkaline, Temperature, and Oxidative Stress. Excepting casein hydrolysis/solubilization surrounding the microbial Cladosporium cladosporioides JA18, all tested strains were ∘ colony. The laccase production was detected on PDA medium able to grow at pH 10. All isolates were able to grow at 4 C containing 5 mM of 2,6 dimethoxyphenol (DMP). Oxidation while only five strains Aspergillus fumigatus JA10, Aspergillus of the substrate is indicated by the appearance of brown color. fumigatiaffinis JA11, Alternaria alternata JA23, Ulocladium Lipase production was tested on PDA medium containing consortiale JA12, and Ulocladium sp. JA17 showed a significant ∘ 10 mL/L of Tween 20 and 0.1 g/L of CaCl2.Positivereaction growth at 45 C. All 21 strains tolerated oxidative stress is accompanied by the presence of precipitates around the generated by 10 mM H2O2 and 500 𝜇M paraquat (Table 3). fungal colony. The enzymes production was expressed as activity ratio (PR) which corresponds to the activity diameter 3.4. Enzymatic Profiling of Isolates. Among the 21 strains (halo of enzymatic reaction) divided by the colony diameter ∘ tested, 13 strains displayed at least one of the five-screened after7daysincubationat30C. activities: protease, amylase, cellulase, lipase, and laccase, in thepresenceof10%NaCl(Table4). Protease and amylase 2.5. Statistical Analysis. The data presented are the average of werethemostabundantactivitiesshownby9and6strains, the results of at least three replicates with a standard error of respectively. Four strains belonging to Cladosporium and less than 10%. Penicillium genera produced laccase while Cladosporium sphaerospermum JA2, Aspergillus flavus JA4, and Engyodon- 3. Results tium album JA7 were able to produce lipase. Cellulase activity was detected only in Penicillium sp. JA15. 3.1. Isolation and Identification of Halotolerant Fungi. Twenty-one fungal isolates were obtained on halophilic 4. Discussion medium containing 10% NaCl and subjected to morphological and molecular identification. Seventeen strains were With regard to bacteria that have been well explored in identified at genus level based on 28S rRNA gene sequences, southern desert region of Tunisia [28–31], data related to while four were identified based on ITS regions. Final fungi are scarce and are limited to truffle and mycorrhiza, so assignment was based on combination of morphological far considered as real specialists of desert environments [32, and 𝛽-tubulin, actin, calmodulin, and glyceraldehyde-3- 33]. Tothe best of our knowledge, this is the first report on the phosphate dehydrogenase genes sequencing (Table 1). The 21 isolation and characterization of fungi from Tunisian desert strains have been identified as Cladosporium cladosporioides and particularly from salt flat. A collection of 21 fungi isolates (𝑛=2), Cladosporium halotolerans (𝑛=1), Cladosporium have been established from samples ashes collected from sphaerospermum (𝑛=2), Alternaria tenuissima (𝑛=1), Sebkha El Melah. These alkalihalotolerant fungi have been Aspergillus flavus (𝑛=1), Aspergillus fumigatiaffinis (𝑛=1), assigned to 15 taxa belonging to 6 genera of Ascomycetes. Aspergillus fumigatus (𝑛=1), Penicillium canescens (𝑛=1), Several studies showed that fungi belonging to Cladosporium, Penicillium chrysogenum (𝑛=3), Penicillium sp. (𝑛=1), Alternaria, and Ulocladium genera were clearly predominant Alternaria alternata (𝑛=3), Ulocladium consortiale (𝑛=1), under desert and salty environments [34, 35]. These fungi Ulocladium sp. (𝑛=1), Engyodontium album (𝑛=1), and have in common thick-walled and strongly melanized spores Embellisia phragmospora (𝑛=1) species. All the strains have which are important for UV, radiation, and desiccation 4 BioMed Research International Link Penzig Sopp KF417564 NCBI Thom Thom (Nees) Wiltshire Cladosporium ITS KF417577 ITS: KF417581 28S KF417559 ITS: KF417578 ITS: KF417585 ITS: KF417583 ITS: KF417582 ITS: KF417579 ITS: KF417580 ITS: KF417584 28S: KF417561 28S: KF417565 28S: KF417562 28S: KF417563 (Emden) E.G. 28S: 28S: KF417560 (Limber) de Hoog Gunde-Cimerman Zalar, de Hoog, and accession number in Penicillium canescens Engyodontium album Alternaria tenuissima Aspergillus flavus Final identification and sphaerospermum Embellisia phragmospora Penicillium chrysogenum Penicillium chrysogenum Cladosporium halotolerans nd nd nd Alternaria alternata Engyodontium album Penicillium chrysogenum Penicillium chrysogenum Embellisia phragmospora Morphological identification Cladosporium cladosporioides/halotolerans ) GPD ( ) ) ) GPD Actin ( -tubulin) -tubulin) ( (Actin) primers 𝛽 𝛽 calmodulin ( ( ( Aspergillus flavus Alternaria tenuissima Alternaria arborescens -tubulin and calmodulin) Penicillium chrysogenum Penicillium chrysogenum 𝛽 Cladosporium halotolerans Alternaria alternata Penicillium canescens group ( Identification based on specific Cladosporium sphaerospermum Alternaria tenuissima similarity Table 1: Identification of fungal isolates. EF568045 JN867470 JN383493 EF669617 (100%) JX997039 (100%) JX997105 (100%) HM214540 (100%) HQ607858(99%) AB572902 (99%) AM176719 (100%) EF568045 (99%) KC009827 (100%) KC009826 (99%) JQ781833 (100%) JX535318 (99%) sp. AM176721 (100%) sp. GU017498 (100%) JX997080 (100%) JN867469 (100%) JX997081 (100%) KC333882 (100%) JQ690087 (100%) JN867468 (100%) chrysogenum chrysogenum . . P. g r i s eof u lv um P. confe r tumP. dip o d omy i s ITS identification Penicillium flavigenum P. commune P Cladosporium Hyalodendron C. sphaerospermum C. cladosporioides Penicillium canescens Aspergillus aureofulgens Penicillium desertorum P Alternaria triticimaculans (100%) A. tenuissima A. mali A. alternata Engyodontium album Cladosporium cladosporioides (100%) C. sphaerospermum C. halotolerans Embellisia phragmospora (100%) nd nd Alternaria Aspergillus Penicillium Cladosporium Cladosporium Penicillium chrysogenum Embellisia/Chalastospora Strain code 28S identification JA1 JA2 JA3 JA4 JA5 JA6 JA7 JA8 JA9 BioMed Research International 5 Penzig sp. sp. KF417572 NCBI Keissler Samson Fresenius Cladosporium Cladosporium Cladosporium ITS: KF417591 ITS: KF417593 ITS: KF417595 ITS: KF417589 ITS: KF417588 ITS: KF417594 ITS: KF417590 ITS: KF417587 ITS: KF417586 28S: KF417574 28S: 28S: KF417573 28S: KF417570 28S: KF417569 cladosporioides cladosporioides 28S: KF417568 28S: KF417566 28S: KF417567 Penicillium Ulocladium ¨ umen) E.G. Simmons Hong, Frisvad, and Alternaria alternata accession number in Aspergillus fumigatus Ulocladium consortiale Final identification and sphaerospermum Aspergillus fumigatiaffinis (Fresenius) G.A. de Vries (Fresenius) G.A. de Vries (Th sp. nd nd Ulocladium Alternaria alternata Penicillium glabrum Aspergillus fumigatiaffinis Morphological identification Cladosporium cladosporioides Cladosporium sphaerospermum Ulocladium tuberculatum/consortiale ) GPD ( ) nd nd nd nd GPD group ( -tubulin) -tubulin) (Actin) primers 𝛽 𝛽 ( ( Alternaria alternata Aspergillus fumigatus Alternaria tenuissima Alternaria arborescens Aspergillus fumigatiaffinis Ulocladium consortiale Identification based on specific Cladosporium sphaerospermum Table 1: Continued. KC009539 JX868638 HQ380770 JN246066 KC167852 (100%) JQ585682 (100%) JQ585682 (100%) HM999943 (99%) JN943567 (99%) HM204457 (99%) GU248332 (98%) FR733874 (99%) HF545316 (99%) KC253955 (99%) JN246066 (99%) JQ781762 (100%) JN942881 (99%) JN107734 (100%) KC009784 (100%) sp. KC139473 (100%) fumigatiaffinis . A. novofumigatus (99%) Davidiella tassiana Aspergillus lentulus A. aff. fumigatus A. fumigatiaffinis Aspergillus aff. fumigatus (100%) A Ulocladium consortiale Cladosporium cladosporioides Penicillium spinulosum P. g l abr um Ulocladium consortiale Alternaria A. arborescens A. alternata ITS identification Alternaria radicina Cladosporium cladosporioides (99%) C. sphaerospermum U. chartarum Cladosporium cladosporioides (100%) nd Alternaria Aspergillus Penicillium Ulocladium Ulocladium Cladosporium Cladosporium Cladosporium/Davidiella Strain code 28S identification JA10 JA11 JA12 JA13 JA14 JA15 JA17 JA18 JA19 6 BioMed Research International NCBI Thom Keissler Keissler ITS: KF417598 ITS: KF417596 ITS: KF417597 28S: KF417575 28S: KF417576 Alternaria alternata Alternaria alternata accession number in Final identification and Penicillium chrysogenum Alternaria alternata Alternaria alternata Penicillium chrysogenum Morphological identification ) ) GPD GPD ( ( -tubulin) primers 𝛽 ( Alternaria alternata Alternaria alternata Alternaria tenuissima Alternaria tenuissima Alternaria arborescens Alternaria arborescens Penicillium chrysogenum Identification based on specific Table 1: Continued. KC341721 (99%) JQ809324 (100%) JX290150 (100%) JX232278 (99%) KC329619 (100%) JN676122 (99%) KC329620 (100%) KC329618 (100%) JX003126 (99%) HQ821479 (100%) P. dip o d omyP. i col r ub a e n s P. commune ITS identification A. porri Penicillium chrysogenum Alternaria brassicae A. tenuissima A. atrans Alternaria alternata A. quercus nd Alternaria Penicillium Strain code 28S identification JA20 JA22 JA23 BioMed Research International 7

Table 2: Effect of salt concentration on fungal growth in solid and liquid media.

Solid media (1) Liquid media (2) Strain code Strain 5% NaCl 10% NaCl 15% NaCl 20% NaCl 5% NaCl 10% NaCl 15% NaCl 20% NaCl JA1 Penicillium chrysogenum 74 72 60 18 83 54 10 0 JA3 Penicillium chrysogenum 100 72 37 0 96 46 11 0 JA22 Penicillium chrysogenum 100 82 41 25 90 46 0 0 AJ5 Penicillium canescens 70 30 20 0 79 44 0 0 JA15 Penicillium sp. 83 70 34 0 53 18 0 0 JA8 Cladosporium halotolerans 80 68 32 18 30 0 0 0 JA2 Cladosporium sphaerospermum 76 64 34 22 47 19 11 0 JA13 Cladosporium sphaerospermum 100 49 25 0 81 79 12 0 JA14 Cladosporium cladosporioides 40 30 10 0 0 0 0 0 JA18 Cladosporium cladosporioides 58 40 24 8 63 61 4 0 JA4 Aspergillus flavus 90 80 48 26 56 16 0 0 JA10 Aspergillus fumigatus 100 76 35 0 100 57 11 0 JA11 Aspergillus fumigatiaffinis 100 46 25 0 52 30 12 0 JA19 Alternaria alternata 52 38 24 0 57 9 0 0 JA20 Alternaria alternata 604000372400 JA23 Alternaria alternata 100 68 20 0 65 12 0 0 JA6 Alternaria tenuissima 100 60 22 0 80 55 17 0 JA9 Embellisia phragmospora 94 50 10 0 78 26 0 0 JA12 Ulocladium consortiale 722800321000 JA17 Ulocladium sp. 100 70 28 0 67 20 0 0 JA7 Engyodontium album 56 36 14 10 43 10 0 0 (1)Relativegrowthonsolidmediaafter7daysincubation=(⌀ colony under salt stress/⌀ colony without salt stress) × 100. (2) Relative growth in liquid media after 7 days incubation = (kinetic curve surface undert sal stress/kinetic curve surface without salt stress) × 100.

Table 3: Effect of alkaline, thermal, and oxidative stresses on fungal growth.

Alkaline stress (1) Thermal stress (2) Oxidative stress (3) Strain code Strain ∘ ∘ [ ] [ 𝜇 ] pH 10 4 C45CH2O2 10 mM Paraquat 500 M JA1 Penicillium chrysogenum 43 39 — 66 74 JA3 Penicillium chrysogenum 42 50 — 84 71 JA22 Penicillium chrysogenum 47 45 — 68 53 JA5 Penicillium canescens 26 28 — 59 63 JA15 Penicillium sp. 43 100 — 100 100 JA8 Cladosporium halotolerans 34 26 — 44 40 JA2 Cladosporium sphaerospermum 21 24 — 52 48 JA13 Cladosporium sphaerospermum 21 43 — 55 44 JA14 Cladosporium cladosporioides 34 38 — 20 31 JA18 Cladosporium cladosporioides — 41 — 18 16 JA4 Aspergillus flavus 46 22 — 47 39 JA10 Aspergillus fumigatus 89 41 61 100 100 JA11 Aspergillus fumigatiaffinis 94 26 100 100 100 JA19 Alternaria alternata 49 35 — 69 89 JA20 Alternaria alternata 58 48 — 100 100 JA23 Alternaria alternata 100 83 40 57 52 JA6 Alternaria tenuissima 57 30 — 81 100 JA9 Embellisia phragmospora 58 67 — 100 100 JA12 Ulocladium consortiale 44 37 36 56 100 JA17 Ulocladium sp. 93 28 100 81 100 JA7 Engyodontium album 34 18 — 66 53 Relative growth of fungal strains under different stresses after 7 days incubation was expressed as follows: (1) (⌀ colony at pH 10/⌀ colony at pH 5) × 100; (2) ⌀ ∘ ∘ ⌀ ∘ × ⌀ ⌀ × ( colony at 45 Cor4C/ colony at 30 C) 100; (3) ( colony with H2O2 or paraquat/ colony without stress) 100. —: not significant growth. 8 BioMed Research International

Table 4: Enzymes activities of fungal isolates in the presence of 10% NaCl.

Strain code Strain Protease Amylase Cellulase Lipase Laccase JA1 Penicillium chrysogenum ++ + −−− JA3 Penicillium chrysogenum ++ −−−− JA22 Penicillium chrysogenum ++−−− AJ5 Penicillium canescens −−−−+ JA15 Penicillium sp. −−+ −− JA8 Cladosporium halotolerans + −−−+ JA2 Cladosporium sphaerospermum +++ −−++ JA13 Cladosporium sphaerospermum − + −−+ JA14 Cladosporium cladosporioides ++−−− JA18 Cladosporium cladosporioides −−−−− JA4 Aspergillus flavus + −−+ − JA10 Aspergillus fumigatus −−−−− JA11 Aspergillus fumigatiaffinis −−−−− JA19 Alternaria alternata −−−−− JA20 Alternaria alternata − + −−− JA23 Alternaria alternata −−−−− JA6 Alternaria tenuissima + −−−− JA9 Embellisia phragmospora −−−−− JA12 Ulocladium consortiale −−−−− JA17 Ulocladium sp. −−−−− JA7 Engyodontium album ++− ++ − AR: activity ratio = (⌀ activity/⌀ colony). −:noactivity;+:AR< 1; ++: 1 < AR < 2; +++: 2 < AR < 3. tolerance [10]. On the other hand, Molitoris et al. [36] at pH 10 and 10% of NaCl. However, the isolates were able reported that other halotolerant and halophilic fungi such to grow when salt was not added to their growing media. as Aspergillus and Cladosporium spp. are predominant in Excepting some Wallemia ichthyophaga the most strictly saline desert soil of Dead Sea. Many Aspergillus species have halophilic fungus [43], all other fungal strains known to been also reported to constitute dominant fungi in desert of dateareabletogrowwithoutsalt,afactconfirmedin Saudi Arabia and Libya [37, 38], and halotolerant species, ourstudy.However,gradualdecreaseinfungalgrowthwas including Aspergillus spp., Penicillium spp., and Cladosporium observed with the increasing of salt concentration in the sphaerospermum, have been consistently isolated from hyper- culture medium. Nineteen strains remain able to grow under saline environments around the globe [39]. In this study, 15% of NaCl, whereas 7 strains were able to tolerate 20% of contrary to many reports on hypersaline environments, no NaCl. This result was confirmed by salt tolerance assay in species belonging to the genera Eurotium, Thrimmatostroma, liquid media as estimated by Biolog system. It is noteworthy Emericella, and Phaeotheca [9]havebeenobtained,probably that fungi were more sensitive to salt stress in liquid media because of the initial alkaline pH of the Sebkha El Melah than in solid media. This could be explained by the alteration salt lake. Actually, the effect of pH on the fungal diversity is of the osmotic gradient, forcing the fungi to expend more controversial. Misra [40] observed that fungal diversity varies energy in the osmoregulatory processes, resulting in slower with the pH while other investigators found no significant growth [44].Moreover,athighersaltconcentrationdeath effectofpHvaluesofwaterandsoilhabitatsonfungal occurs. occurrence [41]. It is more likely that the number of the Regarding the stress of pH, the capacity of the majority of isolated fungi is directly correlated to the organic matter isolates to growth at pH 10 implies firstly that some habitats in contentofwater,mud,andsoilsamples[42]. the salt lake may have a varying pH and secondly that fungi Beside the identification of the recovered fungal isolates can tolerate a wide pH range. Prima facie, the overall results from Sebkha El Melah, the second goal of the current study in solid and liquid media showed that Penicillium chryso- was the detection of some of their physiological and biochem- genum JA1 and JA3, Cladosporium sphaerospermum JA2 and ical features. This allows understanding ecological adaptation JA13, Cladosporium cladosporioides JA18, Aspergillus fumi- to extreme environment and predicts some biotechnological gatus JA10, Aspergillus fumigatiaffinis JA11, and Alternaria usage. The 21 strains have been screened for tolerance to tenuissima JA6 are the most alkalihalotolerant isolates in this extreme NaCl concentrations, basic pH, temperature, and study. ∘ oxidative stress and for the production of important enzy- The tolerance of the strains to extreme 45 Cwastested maticactivitiesinpresenceof10%NaCl. and results indicated that Aspergillus fumigatus JA10, Alter- Excepting Cladosporium cladosporioides JA18, all isolates naria alternata JA23, Ulocladium sp. JA17, and Aspergillus obtained in this study can be considered as moderately fumigatiaffinis JA11 were able to grow. Of particular interest, haloalkaliphilic fungi as deduced from their ability to grow the latter two strains retained 100% of the growth rate BioMed Research International 9 and biomass production as estimated by colony diameter. exposure to high solar radiation. Further studies are needed Moreover, their ability to grow at low temperature may allow in order to elucidate their biogeochemical roles in such an them to better adapt to the big temperature fluctuation in extreme environment and to exploit their promising potential desert environments. Additionally, exposure to substrates to produce new biomolecules such as enzymes and protective generating oxidative stress such as H2O2 at 10 mM and agents against oxidative stress. paraquat at 500 𝜇M did not alter significantly the growth of almost tested strains demonstrating their ability to tolerate oxidative stress. These findings may explain their presence in Conflict of Interests desert regions that are considered amongst the most stressful The authors declare that there is no conflict of interests environments on Earth because of the high UV radiation, regarding the publication of this paper. desiccation,increasedsalinity,lownutrientavailability,seasonal and daily temperature variation, and solar irradiation [6, 10]. Acknowledgments It has been postulated that microorganisms sharing a rich and particular extracellular enzymatic activities are The authors acknowledge the financial support from the common in harsh conditions characterizing their ecological EuropeanUnionintheambitoftheProjectBIODESERT habitat including high level of aridity, temperature, ionic (EU FP7-CSA-SA REGPOT-2008-2, Grant agreement no. strength, and particularly the low nutrient availability [31]. 245746) and the Tunisian Ministry of Higher Education and This implies the need by microorganisms for an effective Scientific Research in the ambit of the laboratory Project LR utilization of each possible available organic compound [45]. MBA20. Atef Jaouani wants to thank the Tunisian Society for Moreover, fungal isolates from hot desert were revealed to Microbial Ecology (ATEM) for supporting publication fees of play an important role in seeds germination by breaking this work. dormancy and increasing water uptake [46]. In the present study, the capacity of fungal isolates to produce extracellular References enzymes was assayed in the presence of 10% of NaCl. Enzymes tested were the following: amylase for degradation [1] J. K. Warren, “Sabkhas, saline mudflats and pans,”in Evaporites: of starch, abundant carbohydrate polymer in many plant Sediments, Resources and Hydrocarbons,pp.139–220,Springer, tissues; protease for degradation of plant and animal proteins; Berlin, Germany, 2006. cellulase which hydrolyses the cellulose, the main component [2] A. A. Gorbushina and W. E. Krumbein, “Rock dwelling fungal ofwood,ubiquitoussubstrateforfungi;andfinallythelaccase communities: diversity of life styles and colony structure,” in involved in plant material delignification and in the synthesis Journey to Diverse Microbial Worlds,J.Seckbach,Ed.,pp.317– of the melanin and related compounds to protect fungi 334, Kluwer Academic Publishers, Amsterdam, The Nether- against radiation. Thirteen strains displayed high productions lands, 2000. at least for one of the five-screened activities while no [3] L. J. Rothschild and R. L. Mancinelli, “Life in extreme environ- clear correlation of enzyme production profile with fungal ments,” Nature,vol.409,no.6823,pp.1092–1101,2001. systematic groups was noted. The abundance of protease [4] S. B. Humayoun, N. Bano, and J. T. Hollibaugh, “Depth distribu- activity is in line with previous data on fungal isolates tion of microbial diversity in mono lake, a meromictic soda lake from extreme environments showing high caseinase activities in California,” Applied and Environmental Microbiology,vol.69, with little effect of salinity and temperature on enzyme no. 2, pp. 1030–1042, 2003. production [36]. The relative limited number of isolates [5] C. Demergasso, E. O. Casamayor, G. Chong, P. Galleguillos, displaying cellulase, amylase, lipase, and laccase activities L. Escudero, and C. Pedros-Ali´ o,“Distributionofprokaryotic´ suggests that high concentration of salt may have an adverse genetic diversity in athalassohaline lakes of the Atacama Desert, effect on enzyme production and/or activity. Their energy was Northern Chile,” FEMS Microbiology Ecology,vol.48,no.1,pp. probably oriented to avoid salt stress due to 10% NaCl rather 57–69, 2004. than the production of bioactive extrolites [47]. However, [6] N. Gunde-Cimerman, A. Oren, and A. Plemenitaˇs, “Adaptation not detecting the enzyme is not absolute confirmation of to life in high salt concentrations,” in Archaea, Bacteria, and anisolateinabilitytoproduceit.Itcouldalsomeanthat Eukarya,p.576,Springer,Dordrecht,TheNetherlands,2005. theenzymewasnotreleasedfromthemyceliumorthat [7]L.Maturrano,F.Santos,R.Rossello-Mora,´ and J. Anton,´ the medium is inadequate for its detection [48]. Laccase “Microbial diversity in Maras salterns, a hypersaline envi- productioninthepresenceof10%ofsaltbytheCladosporium ronment in the Peruvian Andes,” Applied and Environmental group may be of biotechnological interest, for example, in Microbiology,vol.72,no.6,pp.3887–3895,2006. mycoremediation of high salty environments contaminated [8] Q. Wu, G. Zwart, M. Schauer, M. Kamst-van Agterveld, and by organic pollutants. M. Hahn, “Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located In conclusion, fungal community described in this study on the Tibetan Plateau, China,” Applied and Environmental was similar to those reported in inhospitable habitats char Microbiology,vol.72,no.8,pp.5478–5485,2006. acterized by limitation of nutrients, moisture deficit, and [9] C. Gostincar,ˇ M. Grube, S. De Hoog, P. Zalar, and N. Gunde- Cimerman, “Extremotolerance in fungi: evolution on the edge,” FEMS Microbiology Ecology,vol.71,no.1,pp.2–11,2010. 10 BioMed Research International

[10] K. Sterflinger, D. Tesei, and K. Zakharova, “Fungi in hot and [27] M. B. Angelova, S. B. Pashova, B. K. Spasova, S. V. Vassilev, cold deserts with particular reference to microcolonial fungi,” and L. S. Slokoska, “Oxidative stress response of filamentous Fungal Ecology,vol.5,no.4,pp.453–462,2012. fungi induced by hydrogen peroxide and paraquat,” Mycological [11] M. J. Liszka, M. E. Clark, E. Schneider, and D. S. Clark, Research,vol.109,no.2,pp.150–158,2005. “Nature versus nurture: developing enzymes that function [28] A. de Groot, V. Chapon, P. Servant et al., “Deinococcus deserti under extreme conditions,” Annual Review of Chemical and sp. nov., a gamma-radiation-tolerant bacterium isolated from Biomolecular Engineering,vol.3,pp.77–102,2012. the Sahara Desert,” International Journal of Systematic and [12] J. P.Perthuisot, La Sebkha El Melah de Zarzis: genese` et evolution´ Evolutionary Microbiology,vol.55,no.6,pp.2441–2446,2005. d’un bassin salin paralique; Les gisements neolithiques´ des abords [29] A. Chanal, V. Chapon, K. Benzerara et al., “The desert of de la Sebkha el Melah,vol.9ofde Travaux du Laboratoire de Tataouine: an extreme environment that hosts a wide diversity ´ ´ geologie/´ Ecole normale superieure´ , Ecole Normale Superieure,´ of microorganisms and radiotolerant bacteria,” Environmental 1975. Microbiology,vol.8,no.3,pp.514–525,2006. [13] I. cLellan, A. Varela, M. Blahgen et al., “Harmonisation of [30] M. Gtari, D. Daffonchio, and A. Boudabous, “Assessment of physical and chemical methods for soil management in Cork the genetic diversity of Frankia microsymbionts of Elaeagnus Oak forests—lessons from collaborative investigations,” African angustifolia L. plants growing in a Tunisian date-palm oasis by Journal of Environmental Science and Technology,vol.7,no.6, analysis of PCR amplified nifD-K intergenic spacer,” Canadian pp.386–401,2013. Journal of Microbiology,vol.53,no.3,pp.440–445,2007. [14] J. H. Warcup, “The soil-plate method for isolation of fungi from [31] I. Essoussi, F. Ghodhbane-Gtari, H. Amairi et al., “Esterase as soil,” Nature,vol.166,no.4211,pp.117–118,1950. an enzymatic signature of Geodermatophilaceae adaptability [15] K. H. Domsch, W. Gams, and T. H. Anderson, Compendium of to Sahara desert stones and monuments,” Journal of Applied Soil Fungi, Academic Press, London, UK, 1980. Microbiology,vol.108,no.5,pp.1723–1732,2010. [16] J. A. von Arx, The Genera of Fungi Sporulating in Pure Culture, J. Cramer, Vaduz, Germany, 1981. [32] I. Sbissi, M. Neffati, A. Boudabous, C. Murat, and M. Gtari, “Phylogenetic affiliation of the desert truffles Picoa juniperi and [17] E. Kiffer and M. Morelet, Les deuteromyc´ etes:` Classification et Picoa lefebvrei,” Antonie van Leeuwenhoek,vol.98,no.4,pp. cles´ d'identification gen` erique´ , INRA Editions, Paris, France, 429–436, 2010. 1997. [18] D. Liu, S. Coloe, R. Baird, and J. Pedersen, “Rapid mini- [33] I. Sbissi, F. Ghodhbane-Gtari, M. Neffati, H. Ouzari, A. Boud- preparation of fungal DNA for PCR,” Journal of Clinical Micro- abous, and M. Gtari, “Diversity of the desert truffle Terfezia biology,vol.38,no.1,article471,2000. boudieri chatin in southern Tunisia,” Canadian Journal of Microbiology,vol.57,no.7,pp.599–605,2011. [19] T. J. White, T. Bruns, S. Lee, and J. Talor, “Amplification and direct sequencing of fungal ribosomal RNA genes for [34]C.A.Conley,G.Ishkhanova,C.P.McKay,andK.Cullings,“A phylogenetics,” in PCR Protocols: A Guide to Methods and preliminary survey of non-lichenized fungi cultured from the Applications,M.A.Innes,D.H.Gelfand,J.J.Sninsky,andT.J. hyperarid Atacama Desert of Chile,” Astrobiology,vol.6,no.4, White, Eds., pp. 315–322, Academic Press, New York, NY, USA, pp. 521–526, 2006. 1990. [35] E. V. Smolyanyuk and E. N. Bilanenko, “Communities of [20] G. Luo and T. G. Mitchell, “Rapid identification of pathogenic halotolerant micromycetes from the areas of natural salinity,” fungi directly from cultures by using multiplex PCR,” Journal of Microbiology,vol.80,no.6,pp.877–883,2011. Clinical Microbiology,vol.40,no.8,pp.2860–2865,2002. [36] H. P.Molitoris, A. S. Buchalo, I. Kurchenko et al., “Physiological [21] P. Rodrigues, C. Santos, A. Venancio,ˆ and N. Lima, “Species diversity of the fIrst fIlamentous fungi isolated from the hyper- identification of Aspergillus section Flavi isolates from Por- saline Dead Sea,”in Aquatic Mycology Across the Millennium,K. tuguese almonds using phenotypic, including MALDI-TOF D.Hyde,W.H.Ho,andS.B.Pointing,Eds.,vol.5,pp.55–70, ICMS, and molecular approaches,” Journal of Applied Microbi- Fungal Diversity Press, 2000. ology, vol. 111, no. 4, pp. 877–892, 2011. [37] S. I. I. Abdel-Hafez, “Survey of the mycoflora of desert soils in [22] K. Bensch, U. Braun, J. Z. Groenewald, and P. W. Crous, “The Saudi Arabia,” Mycopathologia,vol.80,no.1,pp.3–8,1982. genus cladosporium,” Studies in Mycology,vol.72,pp.1–401, [38] A. H. M. El-Said and A. Saleem, “Ecological and physiological 2012. studies on soil fungi at Western Region, Libya,” Mycobiology, [23] M. L. Berbee, M. Pirseyedi, and S. Hubbard, “Cochliobo- vol. 36, pp. 1–9, 2008. lus phylogenetics and the origin of known, highly virulent pathogens, inferred from ITS and glyceraldehyde-3-phosphate [39]P.Zalar,G.S.deHoog,H.-J.Schroers,P.W.Crous,J.Z.Groe- dehydrogenase gene sequences,” Mycologia,vol.91,no.6,pp. newald, and N. Gunde-Cimerman, “Phylogeny and ecology of 964–977, 1999. the ubiquitous saprobe Cladosporium sphaerospermum,with descriptions of seven new species from hypersaline environ- [24] N. L. Glass and G. C. Donaldson, “Development of primer ments,” Studies in Mycology,vol.58,pp.157–183,2007. sets designed for use with the PCR to amplify conserved genes from filamentous Ascomycetes,” Applied and Environmental [40] J. K. Misra, “Occurrence, distribution and seasonality of aquatic Microbiology,vol.61,no.4,pp.1323–1330,1995. fungiasaffectedbychemicalfactorsinsixalkalinepondsof [25] S.F.Altschul,W.Gish,W.Miller,E.W.Myers,andD.J.Lipman, India,” Hydrobiologia,vol.97,no.2,pp.185–191,1982. “Basic local alignment search tool,” Journal of Molecular Biology, [41] F. T. El-Hissy, S. A. El-Zayat, A. M. Khallil, and M. S. Massoud, vol. 215, no. 3, pp. 403–410, 1990. “Aquatic fungi from the submerged mud of Aswan High Dam [26] H. M. Hassan and I. Fridovich, “Paraquat and Escherichia coli. Lake,” Microbiological Research,vol.152,no.1,pp.27–32,1997. Mechanism of production of extracellular superoxide radical,” [42] A. M. Khallil, F.T. El-Hissy, and E. H. Ali, “Seasonal fluctuations JournalofBiologicalChemistry,vol.254,no.21,pp.10846–10852, of aquatic fungi recovered from Egyptian soil (Delta Region),” 1979. Journal of Basic Microbiology,vol.35,no.2,pp.93–102,1995. BioMed Research International 11

[43] P. B. Matheny, J. A. Gossmann, P. Zalar, T. K. A. Kumar, and D. S. Hibbett, “Resolving the phylogenetic position of the Wallemiomycetes: an enigmatic major lineage of Basidiomy- cota,” Canadian Journal of Botany,vol.84,no.12,pp.1794–1805, 2006. [44] A. Blomberg and L. Adler, “Tolerence of fungi to NaCl,”in Stress Tolerance of Fungi, D. Jennings, Ed., pp. 209–231, 1993. [45] A. Oren, “Bioenergetic aspects of halophilism,” Microbiology and Molecular Biology Reviews,vol.63,no.2,pp.334–348,1999. [46] P. Delgado-Sanchez,´ M. A. Ortega-Amaro, J. F. Jimenez-´ Bremont, and J. Flores, “Are fungi important for breaking seed dormancy in desert species? Experimental evidence in Opuntia streptacantha (Cactaceae),” Plant Biology,vol.13,no.1,pp.154– 159, 2011. [47] J. C. Frisvad, “Halotolerant and halophilic fungi and their extrolite production,” in Adaptation to Life at High Salt Con- centrations in Archaea , Bacteria and Eukarya, N. Gunde- Cimerman, A. Oren, and A. Plemenitaˇs, Eds., pp. 426–440, Springer,Amsterdam,TheNetherlands,2005. [48] A. Abdel-Raheem and C. A. Shearer, “Extracellular enzyme production by freshwater ascomycetes,” Fungal Diversity,vol.11, pp. 1–19, 2002. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 914767, 11 pages http://dx.doi.org/10.1155/2014/914767

Research Article Geodermatophilus poikilotrophi sp. nov.: A Multitolerant Actinomycete Isolated from Dolomitic Marble

Maria del Carmen Montero-Calasanz,1,2 Benjamin Hofner,3 Markus Göker,1 Manfred Rohde,4 Cathrin Spröer,1 Karima Hezbri,5 Maher Gtari,5 Peter Schumann,1 and Hans-Peter Klenk1

1 Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, 38124 Braunschweig, Germany 2 Instituto de Investigacioon´ y Formacioon´ Agraria y Pesquera (IFAPA), Centro Las Torres-Tomejil, Carretera Sevilla-Cazalla de la Sierra, Km 12.2, 41200 AlcaladelR´ ´ıo, Sevilla, Spain 3 Institut fur¨ Medizininformatik, Biometrie und Epidemiologie, Friedrich-Alexander-Universitat¨ Erlangen-Nurnberg,¨ Waldstraße 6, 91054 Erlangen, Germany 4 Helmholtz Centre for Infection Research (HZI), Inhoffenstraße 7, 38124 Braunschweig, Germany 5 Laboratoire Microorganismes et Biomolecules´ Actives, UniversitedeTunisElmanar(FST)etUniversit´ e´ de Carthage (INSAT), 2092 Tunis, Tunisia

Correspondence should be addressed to Maria del Carmen Montero-Calasanz; [email protected] and Hans-Peter Klenk; [email protected]

Received 1 April 2014; Revised 3 June 2014; Accepted 9 June 2014; Published 9 July 2014

Academic Editor: Sara Borin

Copyright © 2014 Maria del Carmen Montero-Calasanz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

A novel Gram-reaction-positive, aerobic actinobacterium, tolerant to mitomycin C, heavy metals, metalloids, hydrogen peroxide, desiccation, and ionizing- and UV-radiation, designated G18T, was isolated from dolomitic marble collected from outcrops in ∘ Samara (Namibia). The growth range was 15–35 C, at pH 5.5–9.5 and in presence of 1% NaCl, forming greenish-black coloured colonies on GYM Streptomyces agar. Chemotaxonomic and molecular characteristics of the isolate matched those described for other representatives of the genus Geodermatophilus. The peptidoglycan contained meso-diaminopimelic acid as diagnostic diaminoacid. The main phospholipids were phosphatidylethanolamine, phosphatidylcholine, phosphatidylinositol, and small amount of diphosphatidylglycerol. MK-9(H4) was the dominant menaquinone and galactose was detected as diagnostic sugar. The major cellular fatty acids were branched-chain saturated acids iso-C16:0 and iso-C15:0 and the unsaturated C17:1𝜔8c and C16:1𝜔7c. The 16S rRNA gene showed 97.4–99.1% sequence identity with the other representatives ofgenus Geodermatophilus.Basedon phenotypic results and 16S rRNA gene sequence analysis, strain G18T is proposed to represent a novel species, Geodermatophilus poikilotrophi.TypestrainisG18T (= DSM 44209T =CCUG63018T). The INSDC accession number is HF970583. The novel R software package lethal was used to compute the lethal doses with confidence intervals resulting from tolerance experiments.

1. Introduction studied for a long time due to difficulties in culturing isolates [5], in spite of the fact that its members are frequently The family cursive was originally proposed by Normand isolated from arid soils [5] and occasionally from arid and et al. [1], but a formal description of the family name was semiarid rock substrates such as rock vanish and marble only published a decade later [2]. At the time of writing, [6, 7], where a variety of environmental changing factors the family comprises the genera Blastococcus, Modestobacter, influence their settlement, growth, and development [8]. and Geodermatophilus (as the type genus). Geodermatophilus Some of them were also isolated from rhizosphere soil [9, 10]. was proposed by Luedemann [3] and was included in the To enable the survival in such extreme ecological niches, Approved Lists of Bacterial Names [4]. This genus was poorly where bacterial cells are suppressed to reactive oxygen species 2 BioMed Research International

∘ (ROS) generating-stresses, those should exhibit a very broad cultivated on GYM Streptomyces medium at 28 C. Colony range of tolerance to multiple and fluctuating environmental features were observed at 4 and 15 days under a binocular stresses, such as solar radiation, desiccation and rehydration, microscope according to Pelczar Jr. [32]. Exponentially grow- temperature fluctuations, salts, and metals [8, 11], and a ing bacterial cultures were observed with an optical micro- probable ionizing-radiation (IR) resistance. The origin of scope (Zeiss AxioScope A1) with a 100-fold magnification this last capability cannot be explained as adaptation to and phase-contrast illumination. Micrographs of bacterial environment, suggesting an “incidental” result of tolerance cells grown on GYM Streptomyces brothafter7dayswere to desiccation, whose DNA damage pattern is similar to that taken with a field-emission scanning electron microscope generated by ionizing radiation in Deinococcus species [12]. (FE-SEM Merlin, Zeiss, Germany). Gram reaction was per- Furthermore, tolerance to hydrogen peroxide and mitomycin formed using the KOH test described by Gregersen [33]. Cell C as indicators of the presence of an efficient microbial motility was observed on modified ISP234 [ ] swarming agar −1 oxidative stress repair and double-strand break repair system, (0.3%, w/v) at pH 7.2 supplemented with (l ) 4.0 g dextrin, characteristics also attributed to radiation resistance, have 4.0 g yeast extract, and 10.0 g malt extract. Oxidase activity been widely studied [13, 14]. Multiple-stress tolerance of the was analysed using filter-paper disks (Sartorius grade 388) 󸀠 󸀠 type strain Geodermatophilus obscurus was already described impregnated with 1% solution of N,N,N ,N -tetramethyl- by Gtari et al. [11], suggesting a correlation between tolerance p-phenylenediamine (Sigma-Aldrich); a positive test was profiles to desiccation, mitomycin C, hydrogen peroxide, and defined by the development of a blue-purple colour after ionizing- and UV-radiation. Previous works of Rainey et al. applying biomass to the filter paper. Catalase activity was [15]andGiongoetal.[16] already revealed the prevalence of determined based on formation of bubbles following the IR resistant Geodermatophilus isolates from arid soil sample addition of 1 drop of 3% H2O2. Growth rates were determined at comparatively the same radiation levels as observed for on plates of GYM Streptomyces medium for temperatures ∘ ∘ Deinococcus species and the predominance of species belongs from 10 to 50 Cat5C increments and for pH values from to the family Geodermatophilaceae detected from intercon- 4.0 to 12.5 (in increments of 0.5 pH units) on modified tinental dust, illustrating, therefore, to resist radiation and ISP2 medium by adding NaOH or HCl, respectively, since desiccation stresses during travel in the high atmosphere. the use of a buffer system inhibited growth of the strains. Fourteen named species have been classified in the The utilization of carbon compounds and acid production ∘ genus Geodermatophilus (ordered by the dates of effective were tested at 28 CusingAPI20NEstrips(bioMerieux)´ publication of the names): G. obscurus [3], G. ruber [9], andGENIIIMicroplatesinanOmnilogdevice(BIOLOG G. nigrescens [17], G. arenarius [18], G. siccatus [19, 20], G. Inc., Hayward, CA, USA) in comparison with the reference saharensis [20, 21], G. tzadiensis [22, 23], G. telluris [24], G. strains G. africanus DSM 45422T, G. amargosae DSM 46136T, soli and G. terrae [10], G. africanus [5, 23], G. normandii G. arenarius DSM 45418T, G. nigrescens DSM 45408T, G. [25], G. taihuensis [26], and G. amargosae [27, 28]. Until now, normandii DSM 45417T, G. obscurus DSM 43160T, G. ruber only the genome of the type strain of the type species, G. DSM 45317T, G. saharensis DSM 45423T, G. siccatus DSM 20T obscurus G- , has been sequenced [29]. Moreover, three 45419T, G. soli DSM 45843T,G.taihuensisDSM 45962T, G. subspecies have been identified and named, but their names telluris DSM 45421T, G. terrae DSM 45844T,andG. tzadiensis 𝑇 were not validly published yet: “G. obscurus subsp. utahensis,” DSM 45416 in parallel assays. The GEN III Microplates were “G. obscurus subsp. dictyosporus”[3], and “G. obscurus subsp. inoculated with cells suspended in a viscous inoculating fluid everesti”[30, 31]. This study describes the taxonomic position (IF C) provided by the manufacturer at a cell density of 70% of a novel species into the genus Geodermatophilus based transmittance (T) for G. amargosae DSM 46136T,at75–79% on a polyphasic approach and its tolerance to different TforG. africanus DSM 45422T,at90%TforG. arenarius environmental stresses. DSM 45418T and G. taihuensis DSM 45962T,andat80–83%T for all other reference strains. Respiration rates (and growth) 2. Materials and Methods were measured yielding a total running time of 5 or 10 days, depending on the strain, in phenotype microarray mode. 2.1. Isolation. During screening for microorganisms from Each strain was studied in two independent repetitions. Data dolomitic marble outcrops in an agriculture area at 1150 masl were exported and analysed using the opm package for R in Samara, near to Namib desert (Namibia), a greenish-black 18T [35, 36] v.1.0.6. Reactions with a distinct behaviour between strain designated as G was isolated (in 1993) and purified the two repetitions were regarded as ambiguous. Clustering as described by Eppard et al. [7]. analyses of the phenotypic microarrays were constructed using the pvclust package for R v.1.2.2. [37]. Enzymatic 2.2. Morphological and Biochemical Characterization. Cul- activities were tested using API ZYM galleries accord- tural characteristics were tested on GYM Streptomyces ing to the instructions of the manufacturer (bioMerieux).´ medium (DSMZ medium 65), TSB agar (DSMZ medium Chemotaxonomic procedures. Whole-cell sugars were pre- 535), GPHF medium (DSMZ medium 553), R2A medium paredaccordingto LechevalierandLechevalier[38], followed (DSMZ medium 830), GEO medium (DSMZ medium 714), by thin layer chromatography (TLC) analysis [39]. Polar PYGV medium (DSMZ medium 621), and Luedemann ∘ lipids were extracted, separated by two-dimensional TLC, medium (DSMZ medium 877) for 15 days at 28 C. To and identified according to procedures outlined by Minnikin determine its morphological characteristics, strain G18T was et al. [40] with modifications proposed by Kroppenstedt and BioMed Research International 3

Goodfellow [41]. Additionally, choline-containing lipids were plates in duplicate in two independent experiments and then −1 −2 detected by spraying with Dragendorff’s reagent (Merck) exposed to a dose of 5–10 J⋅s m in a laminar flow hood [42]. Menaquinones (MK) were extracted from freeze-dried equipped with crossbeam 254 nm UV sources in both side cell material using methanol as described by Collins et al. [43] walls (Safe 2020, Thermo Scientific) for 1, 10, 30, 60, 120, 240, ∘ and analysed by high-performance liquid chromatography and 600 min. After 2 weeks at 28 C, the survival fractions −1 (HPLC) [44]. The extraction and analysis of cellular fatty were calculated based on the c.f.u. mL .TheUVshadow acids were carried out in two independent repetitions from zone was avoided. The tolerance to DNA damaging agent ∘ biomass grown on GYM agar plates held at 28 Cfor4 mitomycin C was tested in two independent experiments days and harvested always from the same sector (the last by incubation of cell suspension at room temperature with −1 quadrant streak). Analysis was conducted using the microbial the antibiotic at a final concentration of 5 𝜇g⋅mL .After identification system (MIDI) Sherlock Version 4.5 (method 1, 5, 10, 20, 40, 60, and 120 min, samples were centrifuged TSBA40, ACTIN6 database) as described by Sasser [45]. The at 3500 rpm for 4 min, washed twice in 0.9% NaCl, and, annotation of the fatty acids in the ACTIN6 peak naming subsequently,seriallydiluted.AliquotswerespreadonGYM table is consistent with IUPAC nomenclature (i.e., double Streptomyces agar in duplicate. After incubation, the survival −1 bond positions identified with reference to the carboxyl fractionswerecalculatedbasedonthec.f.u.mL .Totest groupofthefattyacid),butforconsistencywithother the resistance to hydrogen peroxide, equal volumes of cell publications this has been altered to numbering from the suspensions and 0.5% hydrogen peroxide were incubated at aliphaticendofthemolecule(i.e.,16:1CIS9become16:1 room temperature in two independent experiments. After 1, 𝜔7c, etc.). The composition of peptidoglycan hydrolysates (6 ∘ 5,10,20,40,60,and120min,sampleswerehandledaswas NHCl,100C for 16 h) was examined by TLC as described by previously described in mitomycin experiments to calculate Schleifer and Kandler [46]. All chemotaxonomical analyses the survival fractions. For desiccation tolerance, 25 𝜇Lofcell were conducted under standardized conditions with strain suspension were transferred to individual wells of microtiter T G18 and cultures of the same set of reference strains as listed plates in triplicate. Unsealed microtiter plates were placed in above for morphological and biochemical characterisations. a desiccator (23.5% relative humidity) containing silica gel rubin (Fluka) at room temperature. After 20, 40, 60, 80, and 𝜇 2.3. Genetic and Phylogenetic Analysis. G + C content of 100 days, 250 L of sterile water was added to individual wells chromosomal DNA of strain G18T was determined by HPLC to rehydrate the desiccated cells and then incubated at room according to Mesbah et al. [47]. Genomic DNA extraction, temperature for 1 hour and plated on GYM Streptomyces PCR-mediated amplification of the 16S rRNA gene, and agar. The determination of survival fractions was conducted 18T purification of the PCR product were carried out as described as described above. The sensitivity of strain G to heavy by Rainey et al. [48]. Phylogenetic analysis was based on an metals and metalloids was determined by a growth inhibition alignment of 16S rRNA gene sequences from type strains of plate assay as described by Richards et al. [54]. AgNO3, all species with effectively published names in the Geoder- CuCl2,CoCl2,NiCl2,K2CrO4,Pb(NO3)2,andNa2HAsO4 matophilaceae inferred as described by Montero-Calasanz et were added to GYM Streptomyces medium at 0.1, 0.3, 0.5, 1.0, 2.0, 4.0, 8.0, 10.0, 30.0, and 50.0 mM. Growth was evaluated al. [5]. Pairwise similarities were calculated as recommended ∘ by Meier-Kolthoff et al. [49]. For DNA-DNA hybridization after 1 month at 28 C, determining minimum inhibitory tests, cells were disrupted by using a Constant Systems TS concentration (MIC). 0.75 KW (IUL Instruments, Germany). DNA in the crude lysate was purified by chromatography on hydroxyapatite as 2.5. Statistical Analysis of Tolerance Experiments. To evaluate described by Cashion et al. [50]. DNA-DNA hybridization the tolerance of strain G18T and G. obscurus DSM 43160T was carried out as described by De Ley et al. [51] under with respect to the various physiological challenges, the consideration of the modifications described by Huss et al. median lethal dose (LD50) and the lethal dose 10 (LD10) [52] using a model Cary 100 Bio UV/VIS-spectrophotometer values were computed for both strains. As the number of equipped with a Peltier-thermostatted 6 × 6multicellchanger bacteria initially used in each experiment cannot directly and a temperature controller with in situ temperature probe be obtained and consequently, death rates or survival rates (Varian). cannot be directly computed; standard models based on logistic regression models to obtain LD values are thus not 2.4. Tolerance Experiments. The tolerance of strain G18T available. A negative binomial model for count data [55] and G. obscurus G-20T (DSM 43160), as a positive control was used to estimate of number of survivors dependent on [11], to ionizing- and UV-radiation, mitomycin C, hydro- dose, strain, and experiment. Penalized splines [56], one for gen peroxide, desiccation, and heavy metals/metalloids, was each strain, were used to allow the dose to have a nonlinear assayed using nonsporulating cultures obtained by growth influence on survival fractions. The estimation process was ∘ in TYB medium [53]at28C for 5 days, washed twice with stabilised by using of a square root transformation on dose. 0.9% NaCl, homogenized, and subsequently resuspended in LD50 and LD10 values were subsequently estimated from the saline solution. Ionizing-radiation experiments were carried model and 95% confidence intervals were obtained using a out according to a protocol outlined by Gtari et al. [11]. parametric bootstrap approach [57,Chapter5.4].Detailson To test the resistance to UV-radiation, 0.5 mL aliquots of model fitting and the estimation of the confidence intervals culture suspensions was spread onto GYM Streptomyces agar as well as code to derive LD values from survival count 4 BioMed Research International

acid (cell wall type III), which is consistent with other species of the genus Geodermatophilus [27, 38]. Strain G18T displayed primarily menaquinone MK-9(H4)(82.5%),inagreement with values reported for the family Geodermatophilaceae [2], but also MK-9(H0) (8.8%) and MK-9(H2) (4.8%). Major fatty acids were iso-C16:0 (24.5 ± 0.2%), iso-C15:0 (16.6 ± 1.3%), C17:1𝜔8c (13.9 ± 0.1%) and C16:1𝜔7c (8.3 ± 0.1%), complemented by iso-C16:1 H(5.6 ± 0.9%), anteiso-C15:0 (4.1 ± 0.4%), anteiso-C17:0 (4.4 ± 0.2%), C18:1𝜔9c (3.6 ± 0.1%) and C16:0 (2.4 ± 0.9%). The phospholipids pattern consisted of phosphatidylethanolamine (PE), phosphatidyl- Figure 1: Scanning electron micrograph of strain G18T grown on ∘ choline (PC), phosphatidylinositol (PI), and small amount GYM Streptomyces medium for 7 days at 28 C. of diphosphatidylglycerol (DPG) in accordance with profiles obtained for representatives of other Geodermatophilus species investigated in this study (Table 1). Phosphatidyl- data with one or two strains can be found in the supple- glycerol was not detectable (see Supplementary Figure S3). mentary material (see Figure S4 in Supplementary Material This fact was already predictable based on phospholipids available online at http://dx.doi.org/10.1155/2014/914767). All profiles displayed for other representatives of the genus such computations were done with R [58] using the R software as G. arenarius, G. siccatus, G. tzadiensis, G. normandii, or G. packages mgcv [57]andlethal [59]. amargosae, whose phosphatidylglycerol amounts were nearly imperceptible. Whole-cell sugar analysis revealed galactose 3. Results and Discussion as the diagnostic sugar [38] but also glucose and ribose. Genomic G + C content was 74.4 mol%. 3.1. Morphological and Biochemical Characteristics. Cells of 18T strain G were pleiomorphic and Gram-reaction-positive. 3.3. Molecular Analysis. The almost complete (1514 bp) 16S Individual cells and aggregates were observed, confirming rRNA gene sequence of strain G18T was determined. The 16S reports by Ishiguro and Wolfe [53] of synchronous morpho- rRNA sequence showed the highest degree of similarity with genesis on unspecific media and previous observations on the type strains of G. siccatus (99.1%), G. africanus (99.0%), other representatives of the genus Geodermatophilus [27]. G. amargosae (98.5%), G. normandii (98.4%), G. obscurus In line with the original description by Luedemann [3], (98.3%), G. tzadiensis (98.2%), G. nigrescens (98.1%), G. ruber circular or elliptical motile zoospores and septated filaments (98.0%), and G. arenarius (98.0%). All listed closely related from zoospore germination were observed (Figure 1). Young type strains were placed within the same phylogenetic group colonies were light-red in colour and turned greenish-black at by both, maximum likelihood and maximum-parsimony maturity. Similar colours conversions were already observed estimations (Figure 2). The 16S rRNA gene sequences analysis by Nie et al. [17] and Montero-Calasanz et al. [18, 19, 21, thus strongly supports the assignment of strain G18T to the 22, 25] for type strains of other representatives of the genus, genus Geodermatophilus. However, similarities in 16S rRNA such as G. nigrescens, G. arenarius, G. siccatus, G. saharensis, 18T G. tzadiensis, and G. normandii, when cultivated under the gene sequence between G andsomecloselyrelatedtype same growth conditions (Table 1). Colonies were convex, strains indicated the need to prove the genomic distinctness nearly circular and opaque with a moist surface and an entire ofthetypestrainrepresentingthenovelspeciesbyDNA- 18T 18T DNA hybridization. Strain G displayed a DNA-DNA margin. Strain G grew well on GYM Streptomyces and 35.3 ± 1.0 GEOmediabutdidnotgrowonTSA,R2A,GPHF,PYGV, relatedness of % with the type strain of G. siccatus ∘ 28.1 ± 2.1 and Luedemann media. It grew best at 25–30 Cbutdidnot and %withG. africanus. DNA-DNA hybridizations ∘ ∘ 18T grow below 15 Corabove35C. Growth was observed in of strain G with the type strains of G. amargosae, G. presence of 1% NaCl and between pH 5.5–9.5 (optimal range normandii, G. obscurus, G. tzadiensis, G. nigrescens, G. ruber, pH 7.0–9.5). Results from phenotype microarray analysis and G. arenarius were not conducted, according to Meier- areshownasaheatmapinthesupplementarymaterial Kolthoff et al. [49] that statistically confirmed that the (Figure S1) in comparison to the reference type strains of the threshold value previously established at 97% 16S rRNA gene genus Geodermatophilus. Asummaryofselecteddifferential sequence similarity was too conservative in microbial species phenotypic characteristics is presented in Table 1.Inthe discrimination and determined a Actinobacteria-specific 16S phenotypic clustering significant support> ( 95%) is obtained rRNA threshold at 99.0% with a maximun probability of error for G. poikilotrophi DSM 44209T, G. nigrescens DSM 45408T of 1.00% to get DNA-DNA hybridization values above the 70% threshold recommended by Wayne et al. [60]toconfirm and G. normandii DSM 45417T being most similar to each the species status of novel strains. other regarding the characters present in GEN III Microplates (Suppl. Figure S2). 3.4. Tolerance. Gamma-radiation survival of strain G18T 3.2. Chemotaxonomic Characteristics. Analysis of cell wall (Figure 3(a)) showed not significantly different inactivation components revealed the presence of DL-diaminopimelic kinetic as for G. obscurus DSM 43160T, which is considered BioMed Research International 5 , ;3, ) 4 T .All − − − − − − 16:0 15:0 DSM + − − − − − − T +/ +/ +/ MK- 9(H i-C i-C DPG, PC, PE, PI, PG ydroxyphos- # , , # 8c ) 𝜔 0 − − −− 15:0 16:0 + + ++/ + ++/ ++ −− − −− ), MK- DSM 43160 G. telluris +/ +/ +/ 4 17:1 MK-9 9(H DSM 46136 i-C i-C C PI, PIM DPG, PE, (H Coral pink Black ;9, T , ) 4 − − 16:0 15:0 + ++ ++ + + ++ +++ − +/ +/ MK- black 9(H i-C i-C greenish- G. obscurus DPG, PC, Light-red, PE, PI, PG ;2, G. amargosae ) DSM 45416 T 4 16:0 + −− −− − − − − −− − − MK- 9(H i-C ;15, T DPG, PC, PE, PI, PG † ) ), 0 4 , , 9c 𝜔 −−− −− sp. nov. G18 15:0 16:0 +++++/ −− −− −−− G. tzadiensis +/ 18:1 i-C i-C C MK-9(H MK-9(H ;8, DPG, PME, DSM 45962 PE, PI, 5PL T † ) ), ), 2 0 4 , , − − 15:0 16:0 ++ + + + − − −−−− −−−− −− 17:0 C i-C i-C G. poikilotrophi L, unknown phospholipid; APL, unknown amino-phospholipid; i-, MK-9(H MK-9(H MK-9(H DPG, PME, PE, PI, 3PL DSM 45423 G. taihuensis ) 4 , ;14, T 16:0 15:0 +++ + + − − − Black Light red Light red Black i-C i-C MK-9(H the components are listed in decreasing order of quantity. PI, APL, PG DPG, PC, PE, ∗ G. saharensis ) ), species. Strains: 1, 0 4 ;7, , DSM 45417 T − 16:0 15:0 ++++ + + −−−−−−− − black i-C PI, PG i-C greenish- Light-red, MK-9(H MK-9(H DPG, PC, PE, ) ), 4 4 , , DSM 45419 16:1 − 15:0 16:0 G. normandii +++++ + ++++/ ++ +++++/ ++/ +++++ −− −−− − +/ black PE, PG i-C i-C Geodermatophilus i-H-C Light-red, MK-8(H MK-9(H ;13, DPG, PC, PI, T ) 10% peak area ratio are shown; ), ), G. siccatus 0 4 4 , , 8c ≥ 𝜔 15:0 16:0 ;6, ++++ + ++++++ ++++++ ++++++ ++ +++++/ − − − − T black 17:1 i-C i-C C DPG, PG Light-red, MK-9(H PE, PC, PI, MK-8(H MK-9(H DSM 45422 , ), ), ) 4 4 0 16:0 15:0 ++ ++ − − −− PG (H MK- MK- MK-9 DSM 45418 brown 8(H i-C 9(H i-C PE, PC, DPG, PI, Light-red, ine; PI, phosphatidylinositol; PIM, phosphatidylinositol mannoside; P , G. africanus ) and the type strains of other 4 −− −− −− − −− − −− − − 16:0 15:0 + + ++ ++++++++ ++ −− −− − T +/ +/ +/ MK- black 9(H i-C ;12, i-C DPG, PE, Light-red, T only components making up PC, PI, PG b G. arenarius ]. ) 26 4 , , , , 8c ;5, 𝜔 T −− 15:0 17:0 15:0 16:0 + ++/ ++ ++/ − − −− − − − −−− +/ 17:1 i-C i-C ai-C ai-C C MK-9(H PI, 2PL, PG DSM 45844 DPG, PE, PC, ) ), ), 4 , ambiguous; MK, menaquinones; DPG, diphosphatidylglycerol; PE, phosphatidylethanolamine; PME, phosphatidyl-N-methylethanolamine; PE-OH, h 2 4 , , 8c − DSM 45408 𝜔 −− − − 15:0 16:0 + ++++ −−−− −− +/ +/ 17:1 Black Light-red, red G. terrae PI, PG i-C i-C C MK-8(H MK-9(H MK-9(H DPG, PC, PE, ; 11, T 5% peak area ratio are shown; ) Data taken from Qu et al. [ 4 # , , ≥ 8c ]. 𝜔 15:0 16:0 G. nigrescens −− −− − −− − − −−− − 10 DPG black 17:1 i-C i-C C greenish- Light-red, ;4, MK-9(H PE, PC, PI, T , negative reaction; +/ DSM 45843 − b a ∗ G. soli DSM 45317 ;10, T L-ArginineQuinic acid + + + + L-Histidine L-Alanine Pectin + + D-Gluconic acid + + Glycerol + + L-Rhamnose + D-Arabitol D-Mannitol + + D-Sorbitol + + 1% + + + + + 4% D-Mannose + + Inosine D-Salicin D-Melibiose Stachyose + +/ Turanose Data taken from Jin et al. [ Only components making up Predominant menaquinone(s) NaCl range (w/v) Phospholipids Major fatty acids ColonysurfaceonGYMUtilization of Moist Dry Moist Moist Moist Moist Moist Moist Dry Moist Moist Dry Moist Moist Dry CharacteristicsColony colour on GYM 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Table 1: Differential phenotypic characteristics of strain G18 +, positive reaction; a † phatidylethanolamine; PG, phosphatidylglycerol; PC,iso-branched; phosphatidylchol ai-, anteiso-branched. 45421 G. ruber physiological data are from this study. 6 BioMed Research International

T Geodermatophilus arenarius DSM 45418 (HE654547) 84 67 T / Geodermatophilus telluris DSM 45421 (HE815469) T Geodermatophilus tzadiensis DSM 45416 (HE654545) 45423T 654551 88/75 Geodermatophilus saharensis DSM (HE ) T 79/83 Geodermatophilus amargosae DSM 46136 (HF679056) T 82/66 Geodermatophilus normandii DSM 45417 (HE654546) T Geodermatophilus nigrescens DSM 45408 (JN656711) T Geodermatophilus africanus DSM 45422 (HE654550) T 68/66 Geodermatophilus poikilotrophi G18 (HF970583) 79 73 / 43160T 92356 81/77 Geodermatophilus obscurus DSM (X ) T Geodermatophilus siccatus DSM 45419 (HE654548) T —/64 Geodermatophilus ruber DSM 45317 (EU438905) T 69/85 Geodermatophilus soli DSM 45843 (JN033772) T 100/100 Geodermatophilus taihuensis DSM 45962 (JX294478) T Geodermatophilus terrae DSM 45844 (JN033773) T 73/79 Modestobacter roseus DSM 45764 (JQ819258) T Modestobacter marinus DSM 45201 (EU18 1225) 100/100 T Modestobacter versicolor DSM 16678 (AJ871304) T Modestobacter multiseptatus DSM 44406 (Y18646) T Blastococcus jejuensis DSM 19597 (DQ200983) T Blastococcus aggregatus DSM 4725 (L40614) —/64 T Blastococcus saxobsidens DSM 44509 (FN600641) T Blastococcus endophyticus DSM 45413 (GQ494034) 0.008 Figure 2: Maximum likelihood phylogenetic tree inferred from 16S rRNA gene sequences, showing the phylogenetic position of strain G18T relative to the type strains within the family cursive. The branches are scaled in terms of the expected number of substitutions per site (see size bar). Support values from maximum-likelihood (left) and maximum-parsimony (right) bootstrapping are shown above the branches if equal to or larger than 60%. as highly resistant, according to data reported by Gtari by Montero-Calasanz et al. [22]. Cultures of strain G18T et al. [11]. Strain G18T strains exhibited a shoulder of resis- toleratedanexposuretomitomycinofnearly120minshow- tance similar to D. radiodurans R1 to approximately 5 KGy ing a viability rate of 10%, a value significantly higher than [61], but comparatively lower than the observed one by G. the one observed for the positive control (LD10 = 71 min) T obscurus DSM 43160T. Nevertheless, LD10 of both, G18T (Figure 3(c)). Tolerance of strain G18 (LD10 = 7 min) and G. obscurus DSM 43160T,wasaround9KGy,adose in comparison with the positive control G. obscurus DSM comparatively higher than the displayed one for the high 43160T (LD10 = 8 min) to 0.5% hydrogen peroxide along the radiation resistant strain D. radiodurans R1 [61], although curves did not show any significant differences (Figure 3(d)). other authors reported a LD10 around 10 KGy by using the Based on desiccation survival curves given in Figure 3(e), same strain [62]. UV-radiation survival curves revealed a both strains initially exhibited a similar resistance (LD50). similar progressive loss of viability in both strains during the At the first sample point (20 days), strain G18T showed a first 10 min of exposure until levels below 50%. However, the survival of less than 10%, a value comparatively different to differences between the two resistant phenotypes increased the results observed by G. obscurus DSM 43160T, whose LD10 along the curve, observing a significant variation on viability is reached after 38 days. However, it is worth mentioning at 10% survival (Figure 3(b)). According to radiated doses, that after 110 days a remaining bacterial population of strain strain G18T and G. obscurus DSM 43160T were capable 18T 18T −1 2 G was still observed. Strain G demonstrated thus a to support the lethal effects of 6300–12600 J⋅s m and −1 2 high tolerance to ROS-generating stresses gamma- and UV- 63600–31800 J⋅s m , respectively, sustaining a survival rate radiation, mitomycin C, hydrogen peroxide, and desicca- higher than 10%. Battista [63]andShuklaetal.[62]reported −1 2 tioncomparabletothepositivecontrolG. obscurus DSM LD10 values of 700–1000 J⋅s m for the highly resistant 43160T and, in general terms, to DNA damaging-resistant D. radiodurans R1. The tolerance to UV-radiation in the D. radiodurans R1. This correlative tolerance between ROS- genus Geodermatophilus was already observed, in addition generating stresses was already widely described [11, 62]and to G. obscurus DSM 43160T,inG. tzadiensis DSM 45416T support the hypothesis of efficient and common cellular BioMed Research International 7

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10000 5000 ) −1 1000 20000 500 ) 10000 −1 c.f.u (mL 5000 100 2000 50

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Lethal dose(s) Lethal dose(s) T T T LD50, strain: 43160 LD50, strain: G18T LD50, strain: 43160 LD50, strain: G18 T T LD10, strain: 43160T LD10, strain: G18T LD10, strain: 43160 LD10, strain: G18

95% confidence intervals of lethal doses 95% confidence intervals of lethal doses Strain: 43160T LD10 LD20

LD10 30 T 20 18T LD Strain: 43160 LD Strain: G LD40 T LD30 Strain: G18 LD40 T 50 50 43160 G18T LD LD Strain: - LD60 Strain: 43160T- G18T LD60

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Lethal dose(s) Lethal dose(s) T 50 43160T 50 T LD50, strain: 43160 LD50, strain: G18T LD , strain: LD , strain: G18 T 10 43160T 10 T LD10, strain: 43160 LD10, strain: G18T LD , strain: LD , strain: G18

95% confidence intervals of lethal doses 95% confidence intervals of lethal doses

T LD10 T LD10 Strain: 43160 LD20 Strain: 43160 LD20

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Figure 3: Continued. 8 BioMed Research International

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0 10 20 30 40 50

Time (days) (e)

Figure 3: Estimation of survival following exposure to gamma-radiation (a), UV-radiation (b), mitomycin C (c), hydrogen peroxide (d), and −1 desiccation (e) for strain G18T and G. obscurus DSM 43160T as positive control. The mean c.f.u.mL per strain is given together with the LD50 and LD10 values in the upper panel of each figure; 푦-axis is on a logarithmic scale ((a)–(c), (e)), or on a square root scale (d). The lower panel depicts LD10 and LD50 values per strain and the differences between strains together with confidence intervals. Confidence intervals that do not contain zero (dashed vertical line) indicate significant differences to zero; in case of strain differences this indicates significant differences between strains.

DNA repair mechanisms. Strain G18T showed the highest medium caused an overestimated metals tolerance of strains, 3− 2+ tolerance to AsO4 (MIC = 8.0 mM) followed by Pb justified by the different tolerance range found in both strains 2− 1+ (MIC = 4.0 mM), CrO4 (MIC=4.0mM)andAg (MIC and its mostly correlation with the results described by Gtari =1.0mM).WhereasthegrowthofG. obscurus DSM 43160T et al. [11]. was mainly inhibited by concentrations below 1.0 mM, except Apartfromthephylogeneticanalysisbasedon16SrRNA 3− gene sequences, several phenotypic features support the AsO4 whose sensitivity was 10 times higher (MIC = distinctiveness of strain G18T from representatives of all 80.0 mM) than the one observed for strain G18T (Table 2). It other Geodermatophilus species (Table 1). Based on the has been widely described that the heavy metals/metalloids phenotypic and genotypic data presented, we propose that exposure also produces ROS generation [64]. In this study, a strain G18T represents a novel species within the genus Geo- correspondence with other ROS-generating stresses was not dermatophilus,withthenameGeodermatophilus poikilotrophi observed, in agreement with data reported by Gtari et al. sp. nov. [11]forG. obscurus DSM 43160T,butalsoforModestobacter multiseptatus BC501 and Blastococcus saxobsidens DD2, sug- Description of Geodermatophilus poikilotrophi sp. nov.. Geo- gesting the presence of alternative mechanisms to counteract dermatophilus poikilotrophi (poi.kil.o.troph’i N. L. fem. gen. n. the heavy metals/metalloids stress, such as transport outside poikilotrophi referring to a bacterium that can tolerate diverse the cells [65], adsorption on exocellular structures such as environmental stresses). melanin [66], or enzymatic reduction to less toxic forms [67, Colonies are greenish-black-coloured, circular, and con- 68]. Although it is noteworthy that toxicity levels of lead and vex with a moist surface. Cells are Gram-reaction-positive, copper in G. obscurus DSM 43160T by comparison with the catalase positive, and oxidase negative. No diffusible pig- results displayed by Gtari et al. [11] were much different from ments are produced on any of the tested media. Uti- each other. These divergences in the levels of tolerance might lizes dextrin, D-maltose, D-trehalose, D-cellobiose, sucrose, be due to the differences in the media compositions69 [ ]. In stachyose, D-glucose, D-mannose, D-fructose, D-galactose, addition, it was confirmed that neither phosphate buffer nor L-rhamnose, D-sorbitol, D-mannnitol, myo-inositol, glyc- carbon source concentration present in GYM Streptomyces erol, L-arginine, pectin, D-gluconic acid, quinic acid, methyl BioMed Research International 9

Table 2: Minimum inhibitory concentration of seven heavy metals and metalloids for strain G18T and G. obscurus DSM 43160T.

MIC (mM) of Strain AgNO3 CuCl2 CoCl2 NiCl2 K2CrO4 Pb(NO3)2 NaHAsO4 G18T 1.0 0.1 0.3 0.5 4.0 4.0 8.0 DSM 43160T 0.3 0.1 0.3 0.3 1.0 1.0 80.0

pyruvate, D-lactic acid methyl ester, 훼-ketoglutaric acid, Acknowledgments D-malic acid, bromosuccinic acid, potassium tellurite, Υ- amino-N-butyric acid, acetoacetic acid, propionic acid, The authors would like to acknowledge the help of Bettina acetic acid, as sole carbon source for energy and growth, Straubler¨ and Birgit Grun¨ for DNA-DNA hybridization anal- but not turanose, D-raffinose, D-melibiose, 훽-methyl-D- yses, Gabi Potter¨ for assistance with chemotaxonomy, Brian glucoside, D-salicin, N-acetyl-D-glucosamine, N-acetyl-D- J. Tindall (all at DSMZ, Germany) for his guidance in the galactosamine, N-acetylneuraminic acid, 3-O-methyl-D- chemotaxonomic analyses, and Haitham Sghaier (CNSTN, glucose, D-fucose, inosine, sodium lactate, D- and L- Tunisia) for providing access to the gamma irradiation serine, D-arabitol, D-glucose-6-phosphate, D-aspartic acid, facility. Maria del Carmen Montero-Calasanz is the recipient glycyl-L-proline, L-alanine, L-glutamic acid, L-histidine, L- of a postdoctoral contract from the European Social Fund pyroglutamic acid, L-galactonic acid-훾-lactone, glucuron- Operational Programme (2007–2013) for Andalusia and also amide, mucic acid, D-saccharic acid, p-hydroxyphenylacetic recipient of a DSMZ postdoctoral fellowship (2013–2015). acid, citric acid, 훾-amino-n-butyric acid, and butyric acid. Acid is produced from L-arginine and Υ-amino-N-butyric References acid and can be used as a sole nitrogen source but not N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, N- [1] P.Normand, S. Orso, B. Cournoyer et al., “Molecular phylogeny acetyl-neuraminic acid, D- and L-serine, D-aspartic acid, of the genus Frankia and related genera and emendation of glycyl-L-proline, L-alanine, L-histidine, L-glutamic acid, the family Frankiaceae,” International Journal of Systematic L-histidine, L-pyroglutamic acid, glucuronamide, and 훾- Bacteriology,vol.46,no.1,pp.1–9,1996. amino-n-butyric acid. Positive for aesculin degradation. [2] P.Normand, “Geodermatophilaceae fam. nov., a formal descrip- Negative for reduction of nitrate, denitrification, indole tion,” International Journal of Systematic and Evolutionary production and gelatin degradation. Tests for alkaline Microbiology,vol.56,part10,pp.2277–2278,2006. phosphatase, esterase lipase (C8), esterase (C4), leucine [3] G. M. Luedemann, “Geodermatophilus,anewgenusofthe arylamidase and 훼-glucosidase are positive, but those Dermatophilaceae (Actinomycetales),” Journal of Bacteriology, vol. 96, no. 5, pp. 1848–1858, 1968. for urease, 훽-glucosidase, acid phosphatase, valine arylamidase, Naphthol-AS-BI-phosphohydrolase, lipase (C14), [4]V.B.D.Skerman,V.McGowan,andP.H.A.Sneath,“Approved cystine arylamidase, trypsin, 훼-chymotrypsin, 훼-and훽- lists of bacterial names,” International Journal of Systematic Bacteriology,vol.30,no.1,pp.225–420,1980. galactosidase, 훽-glucuronidase, N-acetyl-훽-glucosamidase, 훼-mannosidase and 훼-fucosidase are negative. Cell growth [5] M. D. Montero-Calasanz, M. Goker,¨ G. Potter¨ et al., “Geo- ∘ dermatophilus africanus sp. nov., a halotolerant actinomycete ranges from 15 to 35 C and from pH 5.5 to 9.5. It is toler- isolated from Saharan desert sand,” Antonie van Leeuwenhoek, ant to gamma- and UV-radiation, mitomycin C, hydrogen 3− vol. 104, no. 2, pp. 207–216, 2013. peroxide, desiccation and heavy metals/metalloids AsO4 , 2+ 2− 1+ [6] C. Urz`ı, L. Brusetti, P.Salamone, C. Sorlini, E. Stackebrandt, and Pb ,CrO4 and Ag . The peptidoglycan in the cell D. Daffonchio, “Biodiversity of Geodermatophilaceae isolated wall contains meso-diaminopimelic acid as diamino acid, from altered stones and monuments in the Mediterranean with galactose as the diagnostic sugar. The predominant basin,” Environmental Microbiology,vol.3,no.7,pp.471–479, menaquinone is MK-9(H4). The main polar lipids are phos- 2001. phatidylethanolamine, phosphatidylcholine, phosphatidyli- [7] M. Eppard, W. E. Krumbein, C. Koch, E. Rhiel, J. T. Staley, and nositol, and small amount of diphosphatidylglycerol. Cellular E. Stackebrandt, “Morphological, physiological, and molecular fatty acids consist mainly of iso-C16:0,iso-C15:0,C17:1휔8c, and characterization of actinomycetes isolated from dry soil, rocks, C16:1휔7c. The type strain has a genomic DNA G + C content of and monument surfaces,” Archives of Microbiology,vol.166,no. 74.4 mol %. The INSDC accession number for the 16S rRNA 1,pp.12–22,1996. gene sequences of the type strain G18T (= DSM 44209T = [8] A. A. Gorbushina, “Life on the rocks,” Environmental Microbi- CCUG 63018T)isHF970583. ology,vol.9,no.7,pp.1613–1631,2007. [9] Y.Q.Zhang,J.Chen,H.Y.Liu,Y.Q.Zhang,W.J.Li,andL.-Y.Yu, “Geodermatophilus ruber sp. nov., isolated from rhizosphere soil of a medicinal plant,” International Journal of Systematic and Conflict of Interests Evolutionary Microbiology, vol. 61, part 1, pp. 190–193, 2011. [10] L. Jin, H.-G. Lee, H.-S. Kim, C.-Y. Ahn, and H.-M. Oh, The authors declare that there is no conflict of interests “Geodermatophilus soli sp. nov. and Geodermatophilus terrae sp. regarding the publication of this paper. nov., two actinobacteria isolated from grass soil,” International 10 BioMed Research International

Journal of Systematic and Evolutionary Microbiology,vol.63, [26] J.-H. Qu, M. Hui, J.-Y. Qu et al., “Geodermatophilus taihuensis part 7, pp. 2625–2629, 2013. sp. nov., isolated from t he interfacial sediment of a eutrophic [11] M. Gtari, I. Essoussi, R. Maaoui et al., “Contrasted resistance of lake,” International Journal of Systematic and Evolutionary stone-dwelling Geodermatophilaceae species to stresses known Microbiology, vol. 63, pp. 4108–4112, 2013. to give rise to reactive oxygen species,” FEMS Microbiology [27] M. C. Montero-Calasanz, M. Goker,¨ M. Rohde et al., “Descrip- Ecology,vol.80,no.3,pp.566–577,2012. tion of Geodermatophilus amargosae sp. nov., to accommodate [12] V.Mattimore and J. R. Battista, “Radioresistance of Deinococcus the not validly named Geodermatophilus obscurus subsp. amar- radiodurans: functions necessary to survive ionizing radiation gosae (Luedemann,1968),” Current Microbiology,vol.68,no.3, are also necessary to survive prolonged desiccation,” Journal of pp.365–371,2014. Bacteriology,vol.178,no.3,pp.633–637,1996. [28]A.OrenandG.M.Garrity,“ValidationListno.158.Listof [13] K. W. Minton, “DNA repair in the extremely radioresistant new names and new combinations previously effectively, but not validly, published,” International Journal of Systematic and bacterium Deinococcus radiodurans,” Molecular Microbiology, vol. 13, no. 1, pp. 9–15, 1994. Evolutionary Microbiology,vol.64,part5,pp.1455–1458,2014. [29] N. Ivanova, J. Sikorski, M. Jando et al., “Complete genome [14] K. W.Minton, “Repair of ionizing-radiation damage in the radi- sequence of Geodermatophilus obscurus type strain (g-20 T),” ation resistant bacterium Deinococcus radiodurans,” Mutation Standards in Genomic Sciences,vol.2,no.2,pp.158–167,2010. Research: DNA Repair,vol.363,no.1,pp.1–7,1996. [30] E. E. Ishiguro and D. W. Fletcher, “Characterization of Geoder- [15] F. A. Rainey, K. Ray, M. Ferreira et al., “Extensive diversity of matophilus strains isolated from high altitude Mount Everest ionizing-radiation-resistant bacteria recovered from Sonoran soils,” Mikrobiologika,vol.12,pp.99–108,1975. Desert soil and description of nine new species of the genus Deinococcus obtained from a single soil sample,” Applied Envi- [31] P. Normand and D. R. Benson, “Genus I. Geodermatophilus ronmmental Microbiology,vol.71,no.9,pp.5225–5235,2005. Luedemann 1968. 1994,” in Bergey's Manual of Systematic Bacteriology: The Actinobacteria,vol.5,part1,pp.528–530, [16] A. Giongo, J. Favet, A. Lapanje et al., “Microbial hitchhikers on Springer,NewYork,NY,USA,2ndedition,2012. intercontinental dust: high-throughput sequencing to catalogue [32] M. J. Pelczar Jr., Manual of Microbiological Methods,McGraw- microbes in small sand samples,” Aerobiologia,vol.29,no.1,pp. Hill,NewYork,NY,USA,1957. 71–84, 2013. [33] T. Gregersen, “Rapid method for distinction of gram negative [17] G. X. Nie, H. Ming, S. Li et al., “Geodermatophilus nigrescens sp. from gram positive bacteria,” European Journal of Applied nov., isolated from a dry-hot valley,” Antonie van Leeuwenhoek, Microbiology,vol.5,no.2,pp.123–127,1978. vol. 101, no. 4, pp. 811–817, 2012. [34] E. B. Shirling and D. Gottlieb, “Methods for characterization [18] M. C. Montero-Calasanz, M. Goker,G.P¨ otter¨ et al., “Geoder- of Streptomyces species,” International Journal of Systematic matophilus arenarius sp.nov.,axerophilicactinomyceteisolated Bacteriology,vol.16,pp.313–340,1966. from Saharan desert sand in Chad,” Extremophiles,vol.16,no. [35] L. A. I. Vaas, J. Sikorski, V. Michael, M. Goker,¨ and H.-P. Klenk, 6, pp. 903–909, 2012. “Visualization and curve-parameter estimation strategies for [19] M. del Carmen Montero-Calasanz, M. Goker,¨ M. Rohde et al., efficient exploration of phenotype microarray kinetics,” PLoS “Geodermatophilus siccatus sp. nov., isolated from arid sand of ONE,vol.7,no.4,ArticleIDe34846,2012. the Saharan desert in Chad,” Antonie van Leeuwenhoek,vol.103, [36] L. A. I. Vaas, J. Sikorski, B. Hofner et al., “Opm: an R package no. 3, pp. 449–456, 2013. for analysing OmniLog (R) phenotype microarray data,” Bioin- [20] J. Euzeby,´ “List of new names and new combinations previously formatics,vol.29,no.14,pp.1823–1824,2013. effectively, but not validly, published,” International Journal of [37] R. Suzuki and H. Shimodaira, “pvclust: hierarchical cluster- Systematic and Evolutionary Microbiology, vol. 63, part 5, pp. ing with p-values via multiscale bootstrap resampling,” R 1577–1580, 2013. package version 01.2-2, http://cran.r-project.org/web/packages/ [21] M. C. Montero-Calasanz, M. Goker,G.P¨ otter¨ et al., “Geoder- pvclust/index.html. matophilus saharensis sp. nov., isolated from sand of the Saharan [38] M. P.Lechevalier and H. A. Lechevalier, “Chemical composition desert in Chad,” Archives of Microbiology,vol.195,no.3,pp.153– as a criterion in the classification of aerobic actinomycetes,” 159, 2013. International Journal of Systematic Bectoriology,vol.20,pp.435– [22] M. D. C. Montero-Calasanz, M. Goker,W.J.Broughtonetal.,¨ 443, 1970. “Geodermatophilus tzadiensis sp. nov., a UV radiation-resistant [39] J. L. Staneck and G. D. Roberts, “Simplified approach to identifi- bacterium isolated from sand of the Saharan desert,” Systematic cation of aerobic actinomycetes by thin layer chromatography,” and Applied Microbiology,vol.36,no.3,pp.177–182,2013. Journal of Applied Microbiology,vol.28,no.2,pp.226–231,1974. [23] A. Oren and G. M. Garrity, “List of new names and new [40] D. E. Minnikin, A. G. O’Donnell, M. Goodfellow et al., “An combinations previously effectively, but not validly, published,” integrated procedure for the extraction of bacterial isoprenoid International Journal of Systematic and Evolutionary Microbiol- quinones and polar lipids,” Journal of Microbiological Methods, ogy,vol.63,pp.3931–3934. vol. 2, no. 5, pp. 233–241, 1984. [24] M. D. C. Montero-Calasanz, M. Goker,¨ G. Potter¨ et al., “Geo- [41] R. M. Kroppenstedt and M. Goodfellow, “The family Ther- dermatophilus telluris sp. nov., an actinomycete isolated from momonosporaceae: Actinocorallia, Actinomadura, Spirillispora Saharan desert sand,” International Journal of Systematic and and Thermomonospora,” i n The Prokaryotes,vol.3ofArchaea Evolutionary Microbiology,vol.63,part6,pp.2254–2259,2013. and Bacteria: Firmicutes, Actinomycetes,pp.682–724,Springer, [25] M. C. Montero-Calasanz, M. Goker,G.P¨ otter¨ et al., “Geoder- New York, NY, USA, 3rd edition, 2006. matophilus normandii sp. nov., isolated from Saharan desert [42] B. J. Tindall, “A comparative study of the lipid composition of sand,” International Journal of Systematic and Evolutionary Halobacterium saccharovorum from various sources,” System- Microbiology,vol.63,part9,pp.3437–3443,2013. atic and Applied Microbiology,vol.13,no.2,pp.128–130,1990. BioMed Research International 11

[43] M. D. Collins, T. Pirouz, M. Goodfellow, and D. E. Min- [61]J.R.Battista,A.M.Earl,andM.J.Park,“WhyisDeinococcus nikin, “Distribution of menaquinones in actinomycetes and radiodurans so resistant to ionizing radiation?” Trends in corynebacteria,” Journal of General Microbiology,vol.100,no. Microbiology,vol.7,no.9,pp.362–365,1999. 2, pp. 221–230, 1977. [62] M. Shukla, R. Chaturvedi, D. Tamhane et al., “Multiple- [44] R. M. Kroppenstedt, “Separation of bacterial menaquinones stress tolerance of ionizing radiation-resistant bacterial isolates by HPLC using reverse phase (RP18) and a silver loaded ion obtained from various habitats: correlation between stresses,” exchanger,” Journal of Liquid Chromatography,vol.5,no.12,pp. Current Microbiology,vol.54,no.2,pp.142–148,2007. 2359–2387, 1982. [63] J. R. Battista, “Against all odds: the survival strategies of [45] M. Sasser, “Identification of bacteria by gas chromatography of Deinococcus radiodurans,” Annual Review of Microbiology,vol. cellular fatty acids,” USFCC Newsl,vol.20,p.16,1990. 51, pp. 203–224, 1997. [46] K. H. Schleifer and O. Kandler, “Peptidoglycan types of bacterial [64] S. J. Stohs and D. Bagchi, “Review article oxidative mechanisms cell walls and their taxonomic implications,” Bacteriological in the toxicity of metals ions,” FreeRadicalBiologyandMedicine, Reviews,vol.36,no.4,pp.407–477,1972. vol. 18, no. 2, pp. 321–336, 1995. [47] M. Mesbah, U. Premachandran, and W. B. Whitman, “Precise [65] D. H. Nies, “The cobalt, zinc, and cadmium efflux system measurement of the G+C content of deoxyribonucleic acid CzcABC from Alcaligenes eutrophus functionsasacation- by high-performance liquid chromatography,” International proton antiporter in Escherichia coli,” Journal of Bacteriology, Journal of Systematic Bacteriology,vol.39,no.2,pp.159–167, vol. 177, no. 10, pp. 2707–2712, 1995. 1989. [66] R. V. Fogarty and J. M. Tobin, “Fungal melanins and their [48] F. A. Rainey, N. Ward-Rainey, R. M. Kroppenstedt, and E. interactions with metals,” Enzyme and Microbial Technology, Stackebrandt, “The genus Nocardiopsis represents a phyloge- vol.19,no.4,pp.311–317,1996. netically coherent taxon and a distinct actinomycete lineage: [67] A. M. Spain, A. D. Peacock, J. D. Istok et al., “Identification proposal of Nocardiopsaceae fam. nov.,” International Journal of and isolation of a Castellaniella species important during Systematic Bacteriology, vol. 46, no. 4, pp. 1088–1092, 1996. biostimulation of an acidic nitrate- and uranium-contaminated [49] J. P. Meier-Kolthoff, M. Goker,¨ C. Sproer,¨ and H. Klenk, aquifer,” Applied and Environmental Microbiology,vol.73,no. “When should a DDH experiment be mandatory in microbial 15, pp. 4892–4904, 2007. taxonomy?” Archives of Microbiology,vol.195,no.6,pp.413–418, 2013. [68] A. C. Poopal and R. S. Laxman, “Studies on biological reduction of chromate by Streptomyces griseus,” Journal of Hazardous [50] P.Cashion, M. A. Holder Franklin, J. McCully, and M. Franklin, Materials,vol.169,no.1–3,pp.539–545,2009. “A rapid method for the base ratio determination of bacterial DNA,” Analytical Biochemistry,vol.81,no.2,pp.461–466,1977. [69] I. V. N. Rathnayake, M. Megharaj, G. S. R. Krishnamurti, N. S. Bolan, and R. Naidu, “Heavy metal toxicity to bacteria: are [51] J. de Ley, H. Cattoir, and A. Reynaerts, “The quantitative the existing growth media accurate enough to determine heavy measurement of DNA hybridization from renaturation rates,” metal toxicity?” Chemosphere,vol.90,no.3,pp.1195–1200,2013. European Journal of Biochemistry,vol.12,no.1,pp.133–142,1970. [52] V. A. R. Huss, H. Festl, and K. H. Schleifer, “Studies on the spectrophotometric determination of DNA hybridization from renaturation rates,” Systematic and Applied Microbiology,vol.4, no. 2, pp. 184–192, 1983. [53] E. E. Ishiguro and R. S. Wolfe, “Induction of morphogenesis in Geodermatophilus by inorganic cations and by organic nitrogenous cations,” Journal of Bacteriology,vol.117,no.1,pp. 189–195, 1974. [54]J.W.Richards,G.D.Krumholz,M.S.Chval,andL.S.Tisa, “Heavy metal resistance patterns of Frankia strains,” Applied and Environmental Microbiology,vol.68,no.2,pp.923–927, 2002. [55] P. McCullagh and J. A. Nelder, Generalized Linear Models, Chapman & Hall, London, UK, 2nd edition, 1989. [56] P. H. C. Eilers and B. D. Marx, “Flexible smoothing with 퐵- splines and penalties,” Statistical Science,vol.11,no.2,pp.89– 121, 1996. [57] S. N. Wood, Generalized Additive Models: An Introduction with R,CRCpress,2006. [58] R Core Team, A Language and Environment for Statistical Computing, R Foundation for Statistical Computing, Vienna, Austria, 2014, http://www.R-project.org/. [59] B. Hofner, “lethal: Compute lethal doses (LD) with confidence intervals,” R package, http://r-forge.r-project.org/projects/lethal/. [60] L. G. Wayne, D. J. Brenner, R. R. Colwell et al., “Report of the Ad Hoc committee on reconciliation of approaches to bacterial systematics,” International Journal of Systematic Bacteriology, vol.37,no.4,pp.463–464,1987. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 480170, 9 pages http://dx.doi.org/10.1155/2014/480170

Research Article Safe-Site Effects on Rhizosphere Bacterial Communities in a High-Altitude Alpine Environment

Sonia Ciccazzo,1 Alfonso Esposito,2 Eleonora Rolli,1 Stefan Zerbe,2 Daniele Daffonchio,1 and Lorenzo Brusetti2

1 DepartmentofFood,EnvironmentalandNutritionalSciences(DeFENS),UniversityofMilan,ViaCeloria2,20133Milan,Italy 2 Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Universita5,39100Bolzano,Italy`

Correspondence should be addressed to Lorenzo Brusetti; [email protected]

Received 2 April 2014; Accepted 14 May 2014; Published 4 June 2014

Academic Editor: George Tsiamis

Copyright © 2014 Sonia Ciccazzo et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The rhizosphere effect on bacterial communities associated with three floristic communities (RW, FI, and M sites) which differed for the developmental stages was studied in a high-altitude alpine ecosystem. RW site was an early developmental stage, FI was an intermediate stage, M was a later more matured stage. The N and C contents in the soils confirmed a different developmental stage with a kind of gradient from the unvegetated bare soil (BS) site through RW, FI up to M site. The floristic communities were composed of 21 pioneer plants belonging to 14 species. Automated ribosomal intergenic spacer analysis showed different bacterial genetic structures per each floristic consortium which differed also from the BS site. When plants of the same species occurred within the same site, almost all their bacterial communities clustered together exhibiting a plant species effect. Unifrac significance value (𝑃 < 0.05) on 16S rRNA gene diversity revealed significant differences (𝑃 < 0.05) between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage FI did not differ significantly from the RWand the M vegetated sites. These results pointed out the effect of different floristic communities rhizospheres on their soil bacterial communities.

1. Introduction can be severely affected by geological dynamics, such as sudden mudslides, alluvial fan sliding, and scree movement, A glacier foreland after glacier retreat can be considered that take back the habitat to an earlier pioneer condition. a cold desert, being composed of habitats characterized Consequently, safe-sites cannot reach the climax but only a by severe climatic regimes and barren substrate with low stable stage of middle maturity [5]. total carbon and nitrogen contents [1]. Rock cracks, concave Furthermore, pioneer plants could select rhizosphere surfaces, and little depressions could ensure protection from microbial communities able to promote plant growth thanks wind, cold, and other harsh environmental conditions [2, 3] totheinteractionsinnutrientcyclingandcarbonsequestra- helping the accumulation of nutrients and the growth of tion [6]. Nevertheless, in a natural ecosystem it is difficult to pioneer plants. Safe-sites are defined as little areas, often assess the effect of vegetation on the rhizosphere bacterial surrounded by big stones, filled up of stone debris or mineral communities, especially in high mountain environments mud [4]. Here, opportunistic pioneer plants could settle characterized by variable environmental parameters (suc- down and form first floristic consortia, significantly affected cessional stage, pH, rainfall, moisture, mineral composition, by the geochemistry of the lytic material. Indeed, physical sampling season, and slope) within a size-limited area typical and biogeochemical weathering processes provide soils of of early and transitional successional stages. The impact soluble nutrients and when the plant colonization on parent of single plants on microbial communities in an alpine materials occurs, the development of glacier foreland into glacier forefield has previously been studied to highlight fertile soils is enhanced by rhizodeposition, root exudation, the relationship between the rhizosphere bacterial commu- decaying biomass, and root mass development. Safe-sites nities of pioneer plants or of the related bare soil and the 2 BioMed Research International

∘ ∘ chronosequence [7–10]. In an early chronosequential stage, in July, 10.3 CinAugust,and8C in September and the mean the rhizosphere microbial community of Poa alpina L. was precipitations were 2.7, 2.5, and 3.6 mm per day, respectively. strongly influenced by the environmental conditions but, in The dominant rock types are schist and gneiss [21]andthe transition and mature stage, plants could select a specific most common soil types are acidic leptosols, regosols, and microbial community [9]. Along a similar chronosequence, umbrisols (mean pH = 4.3) derived from carbonate-free 2 the pioneer plant Leucanthemopsis alpina (L.) Heywood bedrocks. The study site, a foreland of about 3.3 Km left after showed an opposing rhizosphere effect with a specific micro- a quick glacier retreat in the last 160 years [22], was located bial community in the early successional stage only [7]. The above the tree line (2,100 m a.s.l). The analysis of the historical study of the spatial extent of Lc. alpina on the microbial maps of the third Austro-Hungarian topographic survey community and on the physical-chemical parameters in an (the so-called “Franzisco-Josephinische Landesaufnahme”) early successional stage (5, 10 years) did not exhibit significant dated 1850 and the aerial photographs of 1945 and of 2006 trends, supporting the conclusion of Tscherko et al. [9]. orthophotos were helpful to reconstruct the different stages However, in a safe-site, the pioneer vegetation interrelated ofglacierretreat.Thus,comparingthesephotos,oursampling in floristic consortia often exhibited ground stems and root site was ice-free since 1850. tangle with large nets. In this case, a safe-site could be equaled Rhizosphere and soil sampling were carried out in 2011 to a transitional or even a mature grassland for root tangle and May, at the beginning of the plant growing season. Three safe- plant community structure. The floristic community effect in sites (RW,FI, and M sites) characterized by loosely organized such a habitat was observed in natural as well as in artificial assemblages of different plant species and a bare soil (BS site) experimental sites [11]. Osanai et al. [12] demonstrated that were sampled. The sites were less than 20 × 20 cm. RW site, cooccurring plant species from native grassland selected their below an iron rich rock-face, was colonized by Diphasiastrum microbial communities. The effect was generally smaller than alpinum and Gnaphalium supinum L.; FI site, a floristic island forspeciesthatgenerallydonotcooccurnaturally,suchas between big rocks, was colonized by Cladonia sp., Festuca those from agricultural crop systems [13], improved grassland halleri All., Polytrichum sp., Racomitrium sp., Sedum alpestre systems, or fertilized grassland fields [14, 15]. Nunan et al. Vill., and Senecio carniolicus (Willd.) Braun-Blanq.; M site, [16]foundaweakinfluenceofplantcommunityorno a safe-site surrounded by big rocks and characterized by a effect of plant species on the structure and diversity of the flatter area, was colonized by Cetraria islandica (L.) Ach., root-colonizing bacterial community when comparing five Leucanthemopsis alpina (L.) Heywood, Potentilla aurea L., cooccurring grass species from an upland grazed grassland in Rhododendron ferrugineum, Sibbaldia procumbens L., and Scotland. Moreover, topography and other uncharacterized Silene acaulis (L.) Jacq. These sampling sites were carefully environmental factors seemed to be main drivers of the chosen in order to share similar conditions in terms of bacterial community composition. altitude, features, and geology. On the other hand, studies about the effect of plant cover The rhizosphere samples of all the single plant individuals on microbial community in cold environments regarded within a floristic community were collected. Each individual different ecological niches and pointed out the higher sig- plantwascarefullypulledoutthesoil,withoutdamagingits nificance of environmental parameters than the influence single root system. After pulling out each plant and avoiding of the floristic consortia. In Antarctic environments along roots, 4 g of rhizosphere soil strictly adhering to the roots a latitudinal gradient, bacterial diversity of dense vegetation was collected with a pair of sterile tweezers. Three replicates from different locations was comparable whereas bacterial of bulk soil were collected as a control. Moreover, from diversity of “fell-field” vegetation decreased with increasing each safe-site, 50 g of root-free soil was collected and put latitude [17, 18]. In permafrost meadow, steppe, or desert into plastic bags for soil chemical analysis. All the samples steppe, soil characteristics were driving factors of the micro- were immediately transported in refrigerated boxes to the bial diversity [19]. In high elevation arid grassland, a strong laboratory as soon as the logistic constraints permitted and ∘ plant effect was demonstrated for the perennial bunchgrasses they were stored at −80 Cuntilanalysis. Stipa, Hilaria,andfortheinvadingannualgrassBromus [20]. Consequently,theaimofthisworkwastoinvestigateif, 2.2. Soil Chemical Analysis. Soil samples for chemical anal- ∘ in different safe-sites on a deglaciated terrain of the same ysis were oven-dried at 105 C until constant weight and chronosequential age, floristic consortia could select specific then acid was digested (HNO3 concentrated 65% and H2O2 rhizobacterial communities. 30%) in a milestone high performance microwave oven (MLS Mega, Gemini BV Laboratory, Apeldoorn, The Netherlands). 2. Materials and Methods To determine the total organic carbon content, soil samples werealsoacidifiedwithHCl(6M)toeliminateallcarbonates. 2.1. Study Site and Soil Samples. The study site is located in Metals and total phosphorous were determined by induc- ∘ 󸀠 󸀠󸀠 ∘ the upstream subcatchment of Saldur river (46 46 30 N; 10 tively coupled plasma-optical emission spectroscopy (ICP- 󸀠 󸀠󸀠 41 46 E; 2,400 a.s.l.) in the high Matsch valley (South Tyrol, OES, Spectro Ciros CCD, Spectro GmbH, Kleve, Germany). 2 Italy) with a drainage area of 11 km .Themaingeological Nitrogen and C were quantified with an elemental analyzer processes are periglacial and the streamflow is characterized (Flash2000,ThermoScientific).ThepHH2O was measured by the glacier dynamics. During 1970–2000, the valley had using an Accumet AP85 pH (Fisher Scientific Ltd., Pitts- an average rainfall of about 550 mm per year. In 2011, the burgh, PA, USA). To test the level of significance of the ∘ mean temperatures of the plant growing season were 7.3 C observed chemical differences among sites, a Kruskal-Wallis BioMed Research International 3 test was done by using Mann-Whitney pairwise comparison Table 1: Percentage of total nitrogen and carbon content and C/N post hoc test and Bonferroni correction in Past software [23]. ratio in the four safe-sites. Nitrogen % Carbon % C/N 2.3. Molecular Analysis of the Bacterial Communities. Total Safe-site Average St.dev. Average St.dev. Average St.dev. DNA of the rhizosphere and soil samples was extracted using Ultraclean Soil DNA Extraction kit (MO-BIO, Arcore, BS 0.05 0.01 0.62 0.16 11.5 0.61 Italy). Microbial analyses were carried out using denaturing RW 0.27 0.11 3.48 1.47 12.7 0.94 gradient gel electrophoresis (DGGE) [24] to describe the FI 0.72 0.35 10.4 6.03 14.2 1.46 rhizobacterial taxa diversity and automated ribosomal inter- M 0.98 0.85 19.3 18.3 17.5 3.79 genic spacer analysis (ARISA) [25] to describe the structure of the rhizobacterial communities. For DGGE analysis, primers GC357f and 907r were used of an ordination plot to calculate an 𝑅 test statistic on the as described [26]. DGGE was run in a BioRad DCode 0 null hypothesis 𝐻 that there are no differences among universal mutation detection system (Bio-Rad, Milan, Italy). 0 groups. When 𝑅 is near to 0, 𝐻 is true, while when 𝑅 is Polyacrylamide gels were done according to Muyzer et al. 𝐻0 [24].Gelswerestainedfor30minin1xTAEbuffercon- reaching 1, canberejectedandthereisadiscrimination taining SYBR Safe-DNA gel stain (Invitrogen, Milan, Italy). between groups. When ANOSIM statistics approaches 1, Visualization and digital image recording were performed the similarities within groups are larger than the similarity 𝐻0 𝑃 with UVTec (Cambridge, UK). All the visible DGGE bands between groups. We rejected when significance value < were excised and reamplified [24]. Sequencing was per- was 0.05. To test the level of significance between/within formed by STAB-Vida Inc. (Caparica, Portugal). Identifi- plant species ARISA clusters, a Kruskal-Wallis test was done cation of 16S rRNA genes was done by comparison with as above. EMBL/Genebank/DDBJ database and RDP database using The Nexus format of the phylogenetic tree of the DGGE BLASTN and Classifier, respectively. All sequences were identified bands performed by MEGA5 was submitted to submitted to the Ribosomal Database Project (RDP) web the UniFrac web server to test differences among sites server [27] to assign taxonomy. Sequences were submitted based in the UniFrac metric with 100 permutations and the to the Genbank/EMBL/DDBJ databanks under the accession Bonferroni correction factor [31]. A principal coordinates numbers HG763876-HG764130. analysis (PCoA) on the DGGE sequence distance matrix ARISA fingerprint was performed as described by Cardi- for each pair of safe-sites was calculated through UniFrac nale et al. [25] with the ITSF/ITSREub primer set. Denatured metric. On the basis of the DGGE sequences, similar safe- ARISA fragments were run by STAB-Vida Inc. The data sites tended to cluster together. In order to allow a broader were analyzed with Peak Scanner software v1.0 (Applied view of those similarities, the first three principal components Biosystems, Monza, Italy) and a threshold of 40 fluorescent were considered. units was used, corresponding to two times the highest peak detected during the negative control run. Output matrix was 3. Results obtained as in Rees et al. [28]. 3.1. Soil Chemical Analysis. Soil resulted to be a sandy silt soil 2.4. Statistical Analysis of ARISA and DGGE. ARISA matrix with an average texture of 72.3 ± 5.0%ofsand,21.0 ± 4.1% was normalized with the formula (x/∑x)∗1000, where “x” of silt, 6.6 ± 1.3%ofclay,and4.6 ± 1.3%ofhumus;pHwas is the fragment height in units of fluorescence, and then 4.5 ± 0.3%. Average chemical composition of sampled soils transformed on a logarithmic scale for multivariate analysis. was total P 0.7 ± 0.1 mg/kg d.m., total K 7.4 ± 1.0 mg/kg Log-transformationwasusedtostabilizethesamplevariance, d.m., total Ca 3.4 ± 0.6 mg/kg d.m., total Mg 13.4 ± 1.7 mg/kg to reduce the interaction effect, and to normalize the distri- d.m., total Fe 45.4 ± 6.9 mg/kg d.m., and total Al 29.4 ± bution of data. Moreover, log-transformation can combine 5.6 mg/kg d.m. No calcium carbonate was detected. Since the information of a binary matrix with those of a nontrans- those safe-sites were located in proximity of each other, formed data matrix, hence preserving the relative abundance their soil chemical composition did not differ substantially information and down-weighting dominant groups. between sites (Kruskal-Wallis test 𝑃 < 0.05;datanotshown). In order to assess changes in rhizobacterial community Nonitratewasdetected,whileallthenitrogenfoundwas structure between floristic consortia, nonmetric multidimen- represented by ammonia only. Nitrogen increased along an sional scaling (NMDS) was applied with Bray-Curtis algo- ideal gradient from bare soil (0.05% dry weight) to the most rithm. NMDS does not need the assumption of linear associ- vegetated M site (0.98% dry weight) and also total organic ations among variables being described as the most efficient carbon grew up from BS site (0.62% dry weight) to M site ordination method for microbial ecology [29]. Bray-Curtis (19.3% dry weight; Table 1). The trend was confirmed by the is not influenced by recurrent absent values into the matrix, C/N ratio which tended to increase constantly among sites a characteristic very common in ARISA matrices [30]. of more complex vegetative patterns. Bonferroni-corrected ANOSIM (based on Bray-Curtis similarity) was performed Kruskal-Wallis nonparametric analysis of variance showed to test significant differences in the profile composition of significant differences among sites for both total nitrogen, the different sites. ANOSIM is a nonparametric statistical organic carbon content and C/N ratio, except for C and N test, based on permutation, which uses rank similarity matrix content between RW versus FI and RW versus M (𝑃 values 4 BioMed Research International

Table 2: Level of significance (𝑃 values) of the differences in C, Table 3: 𝑃 and 𝑅 values of ANOSIM based on Bray-Curtis similarity N, and C/N content among sites by Bonferroni-corrected Kruskal- of the four safe-sites as grouped after ARISA-NMDS plot analysis. Wallis test. P/R value BS RW FI M CNC/N BS 0.9630 0.9758 0.7937 BS versus RW 0.023 0.023 0.023 RW 0.0124 0.9390 0.7434 BS versus FI 0.028 0.028 0.043 FI 0.0077 0.0005 0.7055 BS versus M 0.019 0.019 0.032 M 0.0092 0.0009 0.0004 RW versus FI 0.175 0.197 0.012 RW versus M 0.772 0.954 0.023 FI versus M 0.004 0.004 0.045 sequences of more than 300 bp were obtained from all sample profiles. RDP facilitated the determination of putative taxonomic affiliation of the recovered sequences. Major bacterial shown in Table 2) explained by a higher standard deviation taxa included Acidobacteria Gp3 and Gp1,Sphingobacteria, of C and N content in M sites. Alphaproteobacteria, Betaproteobacteria, Gammaproteobac- teria, and Actinobacteria (Figure 2). A noteworthy amount of 3.2. Genetic Structure of Bacterial Communities in Alpine uncultured bacteria was found. Shifts in bacterial communi- Bulk Soils and Plant Colonized Safe-Sites. Due to the high ties were visible. Members of the Acidobacteria order were sensitivity of the automated capillary electrophoresis, ARISA present in all the sites samples. They generally represented fingerprints of both rhizosphere and bare soil bacterial com- the most abundant taxon, although a decrease of their relative munities provided complex profiles with peaks ranging from abundance is visible with percentage from BS site (57%) to 151 bp to 1437 bp and the 16S-23S rRNA internal transcribed M site (33%). Proteobacteria were not found in BS site, while spacer region (ITS) richness varied from 43 to 168 peaks. they were scarcely present in RW and FI site rhizospheres The electropherograms, characterized by distinct peaks num- (4%, 8%, resp.). In M site Proteobacteria became more abun- ber and intensity, revealed a shift in bacterial community dant than Acidobacteria (35%). In particular, the increasing structure across the different safe-sites plant communities. abundance of Proteobacteria was due to Alphaproteobacteria, On the NMDS plot (stress value = 0.18), samples from root- being more represented than Gammaproteobacteria and free soil (BS), safe-site of early developmental stage (RW), Betaproteobacteria. A considerable amount of unclassified intermediate stage (FI), and from the most mature one (M) Proteobacteria was also evident in M site. Sphingobacteria showed four separate clusters based on microbial community were recovered with low percentages in RW, FI, and M sites structure (Figure 1). According to axis 1, RW site and BS site rhizospheres whereas members of Actinobacteria taxa were are separated from M and FI sites. According to axis 2, BS and even less abundant being present in FI and M sites rhizo- M sites are separated by RW and FI sites. The unvegetated spheres only. We did not find Sphingobacteria or Actinobac- BS site clustered in a specific group, differentiated by the teria taxa associated with BS samples. According to RDP plant rhizospheres, is clustering closer to the rhizosphere classification, unclassified Acidobacteria or Proteobacteria, as bacterial communities of RW site than to those of FI and well as other uncharacterized bacteria, were quite common M sites. The NMDS separation is partially explained by N within all sites. For example, RW site was almost completely andCcontent,asshownbythosevariablevectors,which colonized by unclassified Acidobacteria and unknown Bacte- influencedmoretheMsitethantheothersafe-sites.ANOSIM ria, except few sequences affiliated to uncultured Burkholde- analysis confirmed a highly significant difference among the ria or to a Chitinophagaceae bacterium. Similarly, FI safe-site four microbial community structures (𝑅 = 0.81; 𝑃 = 0.0001) was mostly colonized by unclassified Acidobacteria, although and the performed test showed significant differences in the more frequent sequences belonging to Bradyrhizobiaceae, pairwise comparisons of the sites with 𝑅 values approaching 1 Chitinophagaceae, and other rarer taxa such as Flavisolibacter in most of the cases (Table 3). Where replicated individuals of sp. or Granulicella sp.werefound.Finally,Msite,themost the same plant species within each safe-site were found, it was differentiated safe-site, counted the presence of unknown possible to denote a plant species effect. This is recognizable Bradyrhizobiaceae, Bradyrhizobium sp., and uncultured Rhi- within RW safe-site, where individuals from D. alpinum and zobiales,aswellasChitinophagaceae, Streptacidiphilus sp., G. supinum formed two clusters significantly different along Thermomonosporaceae, and Xanthomonadaceae. the first axis of NMDS𝑃 ( = 0.032 at the Kruskal-Wallis Despite bias associated with sampling, DNA extraction, test). In FI and M sites the tendency of individuals of the PCR amplification, and DGGE run, the pattern of differ- same species to cluster together seems to disappear, except for ences in bacterial communities composition between the R. ferrugineum, maybe due to the higher number of species unvegetated soils of the BS site and the rhizospheres of interconnected in the safe-site. the three safe-sites was supported by the pairwise UniFrac distance ordinations. Comparing each pair of environments 3.3. Diversity of the Bacterial Communities Associated with using the Bonferroni correction, the UniFrac permutation Alpine Bulk Soils and Pioneer Plants in Safe-Sites. DGGE test significance (𝑃 values < 0.05) showed that the BS site was performed to investigate the different microenviron- samples were significantly different from FI and M sites ments of the three safe-sites and bulk soil in terms of their rhizospheres, but not from RW site rhizospheres. Moreover, dominant bacterial population composition. A total of 255 theFIsiterhizospheredidnotdiffersignificantlyfromM BioMed Research International 5

0.30

MPaur40 0.24

MSaca37 0.18 MRfer16 BS 13 34 MLalp39 0.12 MRfer % MSpro35 C MCisl36 %N 0.06 BS 11

BS 12

−0.3 −0.2FI Clad sp 69 0.1 0.2 0.3 0.4 FI Pol sp 68 −0.06 RW D alp 21 FI S car 64 RWDalp23 62 RW G sup 19 FI Pol sp 66 FIScar67 −0.12FI Rac sp FIFhal65 RWGsup20 FISalp61 RW G sup 18 −0.18 RWDalp22

Figure 1: NMDS plot of the three safe-sites and the bare soil site according to UniFrac distance matrix. BS site was a root-free safe-site, RW site was an early developmental floristic stage, FI site was an intermediate stage, and M site was a later stage. Plant sample names are the following: C isl—Cetraria islandica (L.) Ach.; Clad sp—Cladonia sp.; D alp—Diphasiastrum alpinum;Fhal—Festuca halleri All.; G- sup—Gnaphalium supinum L.; L alp—Leucanthemopsis alpina (L.) Heywood; Pol sp—Polytrichum sp.; P aur—Potentilla aurea L.; Rac sp— Racomitrium sp.; R fer—Rhododendron ferrugineum;Salp—Sedum alpestre Vill.; S car—Senecio carniolicus (Willd.) Braun-Blanq.; S pro— Sibbaldia procumbens L.; S aca—Silene acaulis (L.) Jacq.

100 and RW sites rhizospheres, while the M site rhizospheres 90 exhibited significant differences with the RW site. A PCoA analysis of the UniFrac distance matrix was calculated to 80 assess the overall sequence population similarity among safe- sites (Figure 3). The first axis of PCoA analysis, explaining 70 45.6% of the total variance, showed a shift of the BS site 60 from RW, FI, and M sites. The FI and M communities were located very close together in the same quadrant suggesting %)

( 50 a similar bacterial community composition influenced by variables related to PC1. On the other hand, PC2 (32.6% of 40 the variation) explained the differences between RW site and 30 the other three sites. Finally, the third component (21.8% of thevariance)differentiatedFIfromMandfromBSandRW 20 sites. 10

0 4. Discussion BS RW FI M Safe-sites are defined as environments immediately nearby a Unc. bacteria Alphaproteobacteria pool of seeds, where their germination, growth, and estab- Actinobacteria Sphingobacteria lishment are favorable [4]. In this respect, their availability, Unc. Proteobacteria Acidobacteria Gp1 accessibility, and geomorphological diversity in high moun- Acidobacteria Gp3 Betaproteobacteria tain represent important characteristics of this environment, Gammaproteobacteria since they represent a microsite where a list of ecological Figure 2: Percentage abundance of each taxonomic group for each hazards (snow, wind, frost, and irradiation) are less severe individual rhizobacterial communities of the three safe-sites (RW, than in open terrains and where plant propagules can resist, FI, and M) and the bare soil site (BS) after 16S rRNA gene DGGE- grow, and reproduce. In Matsch valley, belonging to south PCR analysis and band sequencing. Tyrolean Alps, additional ecological hazards are represented 6 BioMed Research International

0.4 0.4

0.3 0.3 M M 0.2 0.2

0.1 BS FI 0.1 BS FI )

0.0 ) 0.0 32.6% ( 32.6% 2 −0.1 ( −0.1 2 PC PC −0.2 −0.2

−0.3 −0.3

−0.4 −0.4 RW −0.5 RW −0.5 −0.6 −0.4 −0.2 0.0 0.2 −0.3 −0.2 −0.1 0.0 0.1 0.2 0.3 0.4 PC1 (45.6%) PC3 (21.8%) (a) (b)

Figure 3: Principal coordinates analysis of the UniFrac distance matrix calculated to assess the overall sequence population similarity among safe-sites. Percentage of variance of the single principal coordinates axis is indicated.

by hot and dry summers, instability of the soil substrate, occurrence of a plant cover effect on the rhizosphere bacterial and excessive animal grazing [32]. Within each safe-site, community within those safe-sites. more than one plant species can grow from seeds, specialized Previous investigations of the rhizosphere effect were vegetative propagules, or plant fragments [33]. In such kind conductedonfewsinglepioneerplantsoringrasslandplots. of environments, pioneer plants tend to grow in very complex Almost all the researches on the rhizosphere effect associated coenosis, where roots are strictly intermixed and interrelated. with a single plant species were achieved on crop or other A great diversity of root exudates from all these plants is plants either in artificial microcosms such as pots or on released in rhizosphere, increasing the carbon amount of agricultural soils such as orchards and crop monocultures. the safe-site. Due to the characteristics of safe-sites, usually Most of these researches demonstrated that peculiar root well isolated among each other by rocks, sand, or mud, exudation and rhizodeposition of different plant species an analysis to understand the occurrence of a vegetation could select the structural and functional diversity of the effect on rhizobacterial communities cannot be done with associated rhizosphere bacterial communities [34–36]. On traditional squared-plots, where more safe-sites are sampled theotherhand,aconsistentnumberofstudieshaveshowed smoothing possible differences between them. Hence, we that several environmental parameters, that is, soil type, decided to study three kinds of safe-sites at different stages soil characteristics, growth stage, management practices, of morphological development, by sampling each single and growing season may influence the composition of the rhizospherefromallthegrowingplantindividuals. microbial communities in the rhizosphere [37–44]. Past The vegetation complexity of the three safe-sites (RW, studies about a natural alpine ecosystem investigated single FI, and M) raised from a simple colonization of two species plant species along successional chronosequences and found (RW site) to the colonization of lichens, mosses, and few inconsistent effects of pioneer plants on rhizosphere micro- herbaceous plant species (FI site) till M site, where five bial communities. For example, while the rhizobacterial herbaceous species and one woody species (R. ferrugineum) community of Lc. alpina was different from the interspace were found. We discovered a distinct clustering of bacterial community in an early successional chronosequential stage, communities according to RW, FI, and M vegetation types inalaterstageitbecamesimilartotheinterspacecommunity. that are significantly diverse from the unvegetated soil (BS Inthiscase,itseemedthattheinfluenceofLc. alpina site). We also found that a gradient in terms of C and N depended on soil age and that nutrient availability could enrichments from BS site to the most developed M site was influence the bacterial community structure7 [ ]. In another an important determinant of microbial community profiles. study case, Lc. alpina individuals in the early successional UniFrac analysis showed site-shifts in bacterial diversity stage (5, 10 years) of a glacier forefield showed no selective which suggest a specialized physiology adapted to the pecu- effect on the microbial community, since a similar bacterial liar site environmental conditions. Moreover, the differences community structure was apparent up to 40 cm of distance among safe-sites, according to C and N gradients, support the to the plant [8]. Another single pioneer plant, P. alpina , BioMed Research International 7 did not exhibit a selective role on its rhizosphere bacterial the intermediate plant colonization stage, FI site, did not community in the pioneer stage of a chronosequence, maybe differ significantly from the RW and the M vegetated sites. due to the harsh environmental conditions of the plot where Previous studies [9, 45, 46] showed that the development it was growing [9]. However, by investigating a more mature of the soil microbial community in alpine glaciers was soil, the same plant species could select a specific microbial determined by the accumulation of soil TOC and total community but related to soil properties and carbon supply. nitrogen. The increasing content of C and N in the floristic On the other hand, safe-sites are more complex than consortia corresponded with increased floristic develop- single pioneer plant individuals in a cold environment, mental stage. Soil nutrients and C influenced the bacterial but they show less complexity than a homogeneous plot community composition along a chronosequence [7], while carefully designed in mountain grasslands. Real safe-sites in the Mendenhall glacier chronosequence [47]theywere are much less homogeneous, being shaped by the history of not correlated with the rhizobacterial communities. These the microarea where they are such as dynamical differences different conclusions seem to strongly depend on the adopted in climate, in geophysical features, or in biota colonization experimental design. Cultural-independent techniques based which determine complicated patterns and often unique rates on phospholipid fatty acid (PLFA) determination [9, 10], to of soil development [1]. In our case, due to the quick glacier point out the different concentration of bacterial/fungal fatty melting in the last 80 years, the 160-year soil represents the acids and to compare the Gram-positives/Gram-negatives only transitional step of the glacier moraine between earliest ratio, or molecular methods like restriction fragment length stages (<10 years) and mature soil (>500 years). As shown polymorphism (RFLP) and DGGE analyses [7, 8]couldnot by aerial photos, orthophotos, and a topographic survey, one have enough resolution to detect little changes in the bacterial of the glacier tongues of the Weisskugel glacier has been community genetic structure due to faint environmental retreating with a discontinuous movement. Consequently, variables [48]. The ARISA analysis we used, targeting the there was no constant gradient of soil age but distinct block intergenic 16S-23S rRNA gene highly variable ITS region, stages where soil age is invariable. In this sense, the 160-year- showed more sensitivity and enabled the detection up to old stage is more stable than an earlier successional soil and subspecies level, increasing the chance of the analysis to it can host a larger number of plant species. Nevertheless it detect very little effects on complex bacterial communities was possible to distinguish hundreds of safe-sites of which [49]. the three chosen were the most represented. Within the stable block stage of 160 years old, the measured differences in rhizobacterial composition and soil parameters supported 5. Conclusions the hypothesis that the plant community composition of each floristic consortium exhibited an effect on the rhizobacterial Despite the harsh environmental condition of the natural communities widely documented in studies done in quite alpine ecosystem and the tight complex root system of different ecosystems. For example, Nunan et al. [16] demon- the safe-site, our results support the capability of different strated a more important influence of the plant community pioneer plant consortia to select specific rhizobacterial com- composition than of the individual plant species on the munities with an increase of bacterial diversity according to root colonizing bacterial community in an upland grazed the increase of soil maturation. Moreover, when plants of grassland, whereas Osanai et al. [12]showedasignificant thesamespeciesoccurredinthesamesite,theassociated impact of the plant species on the soil bacterial community rhizobacterial communities clustered more strictly together composition. Similar results were obtained comparing the according to their genetic structures, confirming the high rhizosphere bacterial communities of three plant species of similarity of the rhizobacterial communities within individ- an arid grassland [20]. ualsofthesamepioneerplantspecies. The rhizosphere bacterial communities of RW site, characterized by only two different plant species, clustered more closely with the BS site than with the vegetated ones showing Conflict of Interests a simpler bacterial community, as confirmed by the UniFrac The authors declare that there is no conflict of interests analysis which detected no significant difference between the regarding the publication of this paper. two sites. Although FI and M sites had a similarity of about 56%,insidetheFIsitewerefoundrhizobacterialcommunities of mosses and lichens which did not cluster strictly with Acknowledgments theplantones.Thepresenceoflichensandmossesinthe same site could explain why the bacterial community of This research was financed by the Dr. Erich-Ritter and the theFIsiterepresentedanintermediatestagebetweenthe Dr. Herzog-Sellenberg Foundation within the Stifterverband RW site and the M site. The M site, colonized by individ- fur¨ die Deutsche Wissenschaft, Project “EMERGE: retreating uals of six plant species, could be considered a later stage glaciers and emerging ecosystems in the Southern Alps” where floristic consortia selected a more complex bacterial (CUP n. I41J11000490007). Partial funds came from the Free community which significantly differs from the one of BS University of Bozen/Bolzano internal funds TN5026 “Effects andRWsites.TheUniFracanalysisshowedthattheBS of climate change on high-altitude ecosystems” (CUP n. communities were distinct from ones of the FI and M sites I41J10000960005). The authors would like to thank Elisa and were weakly similar to the ones of RW site. Moreover, Varolo for plant species identification. 8 BioMed Research International

References [17]E.Yergeau,S.Bokhorst,A.H.L.Huiskes,H.T.S.Boschker,R. Aerts,andG.A.Kowalchuk,“Sizeandstructureofbacterial, [1] J. A. Matthews, The Ecology of Recently-Deglaciated Terrain: A fungal and nematode communities along an Antarctic environ- Geoecological Approach to Glacier Forelands and Primary Suc- mental gradient,” FEMS Microbiology Ecology,vol.59,no.2,pp. cession, Cambridge University Press, Cambridge, UK, 1992. 436–451, 2007. [2]A.Jumpponen,H.Vare,¨ K. G. Mattson, R. Ohtonen, and J. M. [18] E. Yergeau, K. K. Newsham, D. A. Pearce, and G. A. Kowalchuk, Trappe, “Characterization of “safe sites” for pioneers in primary “Patterns of bacterial diversity across a range of Antarctic succession on recently deglaciated terrain,” Journal of Ecology, terrestrial habitats,” Environmental Microbiology,vol.9,no.11, vol. 87, no. 1, pp. 98–105, 1999. pp. 2670–2682, 2007. [3] K. Mizuno, “Succession processes of alpine vegetation in [19]X.F.Zhang,L.Zhao,S.J.XuJr.,Y.Z.Liu,H.Y.Liu,andG.D. response to glacial fluctuations of tyndall glacier, Mt. Kenya, Cheng, “Soil moisture effect on bacterial and fungal community Kenya,” Arctic and Alpine Research,vol.30,no.4,pp.340–348, in Beilu River (Tibetan Plateau) permafrost soils with different 1998. vegetation types,” Journal of Applied Microbiology,vol.114,no. [4] J.L.Harper,J.N.Clatworthy,I.H.McNaughton,andG.S.Sagar, 4, pp. 1054–1065, 2013. “The evolution of closely related species living in the same area,” [20] C. R. Kuske, L. O. Ticknor, M. E. Miller et al., “Comparison Evolution,vol.15,no.2,pp.209–227,1961. of soil bacterial communities in rhizospheres of three plant [5] S. Pignatti, Ecologia del Paesaggio, U.T.E.T, Milan, Italy, 1994. species and the interspaces in an arid grassland,” Applied and [6]R.Hayat,S.Ali,U.Amara,R.Khalid,andI.Ahmed,“Soil Environmental Microbiology,vol.68,no.4,pp.1854–1863,2002. beneficial bacteria and their role in plant growth promotion: a [21] G. Habler, M. Thoni,¨ and B. Grasemann, “Cretaceous meta- review,” Annals of Microbiology, vol. 60, no. 4, pp. 579–598, 2010. morphism in the Austroalpine Matsch Unit (Eastern Alps): the [7] I.P.Edwards,H.Burgmann,¨ C. Miniaci, and J. Zeyer, “Variation interrelation between deformation and chemical equilibration in microbial community composition and culturability in the processes,” Mineralogy and Petrology,vol.97,no.3-4,pp.149– rhizosphere of Leucanthemopsis alpina (L.) Heywood and 171, 2009. adjacent bare soil along an alpine chronosequence,” Microbial [22] C. Knoll and H. Kerschner, “A glacier inventory for South Ecology,vol.52,no.4,pp.679–692,2006. Tyrol, Italy, based on airborne laser-scanner data,” Annals of [8]C.Miniaci,M.Bunge,L.Duc,I.Edwards,H.Burgmann,¨ and J. Glaciology,vol.50,no.53,pp.46–52,2009. Zeyer, “Effects of pioneering plants on microbial structures and [23] Ø.Hammer,D.A.T.Harper,andP.D.Ryan,“Past:paleontolog- functions in a glacier forefield,” Biology and Fertility of Soils,vol. ical statistics software package for education and data analysis,” 44, no. 2, pp. 289–297, 2007. Palaeontologia Electronica,vol.4,no.1,pp.1–9,2001. [9] D. Tscherko, U. Hammesfahr, M.-C. Marx, and E. Kandeler, [24] G. Muyzer, E. C. de Waal, and A. G. Uitterlinden, “Profiling “Shifts in rhizosphere microbial communities and enzyme of complex microbial populations by denaturing gradient gel activity of Poa alpina across an alpine chronosequence,” Soil electrophoresis analysis of polymerase chain reaction-amplified Biology and Biochemistry, vol. 36, no. 10, pp. 1685–1698, 2004. genescodingfor16SrRNA,”AppliedandEnvironmentalMicro- [10] D. Tscherko, U. Hammesfahr, G. Zeltner, E. Kandeler, and R. biology,vol.59,no.3,pp.695–700,1993. Bocker,¨ “Plant succession and rhizosphere microbial commu- [25] M. Cardinale, L. Brusetti, P. Quatrini et al., “Comparison of nities in a recently deglaciated alpine terrain,” Basic and Applied different primer sets for use in automated ribosomal intergenic Ecology,vol.6,no.4,pp.367–383,2005. spacer analysis of complex bacterial communities,” Applied and [11] F. E. Z. Haichar, C. Marol, O. Berge et al., “Plant host habitat and Environmental Microbiology,vol.70,no.10,pp.6147–6156,2004. root exudates shape soil bacterial community structure,” ISME [26]A.M.Sass,H.Sass,M.J.L.Coolen,H.Cypionka,andJ. Journal,vol.2,no.12,pp.1221–1230,2008. Overmann, “Microbial communities in the chemocline of a [12]Y.Osanai,D.S.Bougoure,H.L.Hayden,andM.J.Hovenden, hypersaline deep-sea basin (Urania basin, Mediterranean Sea),” “Co-occurring grass species differ in their associated microbial AppliedandEnvironmentalMicrobiology,vol.67,no.12,pp. community composition in a temperate native grassland,” Plant 5392–5402, 2001. and Soil,vol.368,no.1-2,pp.419–431,2013. [27] J.R.Cole,Q.Wang,E.Cardenasetal.,“TheRibosomalDatabase [13]R.Costa,M.Gotz,¨ N. Mrotzek, J. Lottmann, G. Berg, and Project: improved alignments and new tools for rRNA analysis,” K. Smalla, “Effects of site and plant species on rhizosphere Nucleic Acids Research,vol.37,no.1,pp.D141–D145,2009. community structure as revealed by molecular analysis of [28] G. N. Rees, D. S. Baldwin, G. O. Watson, S. Perryman, and D. microbial guilds,” FEMS Microbiology Ecology,vol.56,no.2,pp. L. Nielsen, “Ordination and significance testing of microbial 236–249, 2006. community composition derived from terminal restriction [14] E. Benizri and B. Amiaud, “Relationship between plants and soil fragment length polymorphisms: application of multivariate microbial communities in fertilized grasslands,” Soil Biology and statistics,” Antonie van Leeuwenhoek,vol.86,no.4,pp.339–347, Biochemistry,vol.37,no.11,pp.2055–2064,2005. 2004. [15]A.K.Patra,L.Abbadie,A.Clays-Josserandetal.,“Effectsof [29] K. R. Clarke, “Non-parametric multivariate analyses of changes management regime and plant species on the enzyme activity in community structure,” Australian Journal of Ecology,vol.18, and genetic structure of N-fixing, denitrifying and nitrifying no. 1, pp. 117–143, 1993. bacterial communities in grassland soils,” Environmental Micro- [30] K. R. Clarke and R. M. Warwick, Change in Marine Commu- biology,vol.8,no.6,pp.1005–1016,2006. nities:AnApproachtoStatisticalAnalysisandInterpretation, [16] N. Nunan, T. J. Daniell, B. K. Singh, A. Papert, J. W. McNicol, PRIMER-E, Plymouth, UK, 2nd edition, 2001. and J. I. Prosser, “Links between plant and rhizoplane bacterial [31] M. Hamady, C. Lozupone, and R. Knight, “Fast UniFrac: communities in grassland soils, characterized using molecular facilitating high-throughput phylogenetic analyses of microbial techniques,” AppliedandEnvironmentalMicrobiology,vol.71, communities including analysis of pyrosequencing and Phy- no. 11, pp. 6784–6792, 2005. loChip data,” ISME Journal,vol.4,no.1,pp.17–27,2010. BioMed Research International 9

[32] K. M. Urbanska, Safe Sites—Interface of Plant Population Ecol- [47] J. E. Knelman, T. M. Legg, S. P. O’Neill et al., “Bacterial ogy and Restoration Ecology. Restoration Ecology and Sustainable community structure and function change in association with Development, Cambridge University Press, Cambridge, UK, colonizer plants during early primary succession in a glacier 1997. forefield,” Soil Biology and Biochemistry,vol.46,pp.172–180, [33] R. Chou, C. Vardy, and R. L. Jefferies, “Establishment from 2012. leaves and other plant fragments produced by the foraging [48] R. Danovaro, G. M. Luna, A. Dell’Anno, and B. Pietrangeli, activites of geese,” Functional Ecology,vol.6,no.3,pp.297–301, “Comparison of two fingerprinting techniques, terminal 1992. restriction fragment length polymorphism and automated [34] H. P. Bais, S.-W. Park, T. L. Weir, R. M. Callaway, and J. M. ribosomal intergenic spacer analysis, for determination of Vivanco, “How plants communicate using the underground bacterial diversity in aquatic environments,” Applied and information superhighway,” Trends in Plant Science,vol.9,no. Environmental Microbiology,vol.72,no.9,pp.5982–5989, 1,pp.26–32,2004. 2006. [35] J.Farrar,M.Hawes,D.Jones,andS.Lindow,“Howrootscontrol [49] A. Okubo and S.-I. Sugiyama, “Comparison of molecular fin- the flux of carbon to the rhizosphere,” Ecology,vol.84,no.4,pp. gerprinting methods for analysis of soil microbial community 827–837, 2003. structure,” Ecological Research,vol.24,no.6,pp.1399–1405, 2009. [36]A.M.Hirsch,W.D.Bauer,D.M.Bird,J.Cullimore,B.Tyler, andJ.I.Yoder,“Molecularsignalsandreceptors:controlling rhizosphere interactions between plants and other organisms,” Ecology,vol.84,no.4,pp.858–868,2003. [37]C.M.Hansel,S.Fendorf,P.M.Jardine,andC.A.Francis, “Changes in bacterial and archaeal community structure and functional diversity along a geochemically variable soil profile,” AppliedandEnvironmentalMicrobiology,vol.74,no.5,pp. 1620–1633, 2008. [38] N. M. Kennedy, D. E. Gleeson, J. Connolly, and N. J. W.Clipson, “Seasonal and management influences on bacterial community structureinanuplandgrasslandsoil,”FEMS Microbiology Ecology,vol.53,no.3,pp.329–337,2005. [39] G. A. Kowalchuk, D. S. Buma, W. de Boer, P. G. L. Klinkhamer, and J. A. van Veen, “Effects of above-ground plant species composition and diversity on the diversity of soil-borne microorganisms,” Antonie van Leeuwenhoek,vol.81,no.1–4,pp.509– 520, 2002. [40] C. L. Lauber, M. S. Strickland, M. A. Bradford, and N. Fierer, “The influence of soil properties on the structure of bacterial and fungal communities across land-use types,” Soil Biology and Biochemistry,vol.40,no.9,pp.2407–2415,2008. [41] P. Marschner, C.-H. Yang, R. Lieberei, and D. E. Crowley, “Soil and plant specific effects on bacterial community composition in the rhizosphere,” Soil Biology and Biochemistry,vol.33,no.11, pp.1437–1445,2001. [42]M.E.Schutter,J.M.Sandeno,andR.P.Dick,“Seasonal,soil type, and alternative management influences on microbial communities of vegetable cropping systems,” Biology and Fertility of Soils,vol.34,no.6,pp.397–410,2001. [43] N. Weinert, Y. Piceno, G.-C. Ding et al., “PhyloChip hybridization uncovered an enormous bacterial diversity in the rhizosphere of different potato cultivars: many common and few cultivar-dependent taxa,” FEMS Microbiology Ecology,vol.75, no. 3, pp. 497–506, 2011. [44]T.Wu,D.O.Chellemi,J.H.Graham,K.J.Martin,andE.N. Rosskopf, “Comparison of soil bacterial communities under diverse agricultural land management and crop production practices,” Microbial Ecology,vol.55,no.2,pp.293–310,2008. [45] D. A. Lipson, S. K. Schmidt, and R. K. Monson, “Links between microbial population dynamics and nitrogen availability in an alpine ecosystem,” Ecology,vol.80,no.5,pp.1623–1631,1999. [46] R. Ohtonen, H. Fritze, T. Pennanen, A. Jumpponen, and J. Trappe, “Ecosystem properties and microbial community changes in primary succession on a glacier forefront,” Oecologia, vol. 119, no. 2, pp. 239–246, 1999. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 568549, 8 pages http://dx.doi.org/10.1155/2014/568549

Research Article Contrasted Reactivity to Oxygen Tensions in Frankia sp. Strain CcI3 throughout Nitrogen Fixation and Assimilation

Faten Ghodhbane-Gtari,1,2 Karima Hezbri,1 Amir Ktari,1 Imed Sbissi,1 Nicholas Beauchemin,2 Maher Gtari,1,2 and Louis S. Tisa2

1 Laboratoire Microorganismes et Biomolecules´ Actives, UniversiteTunisElManar(FST)andUniversit´ e´ Carthage (INSAT), Campus Universitaire, 2092 Tunis, Tunisia 2 Department of Molecular, Cellular & Biomedical Sciences, University of New Hampshire, 46 College Road, Durham, NH 03824-2617, USA

Correspondence should be addressed to Louis S. Tisa; [email protected]

Received 18 April 2014; Revised 28 April 2014; Accepted 15 May 2014; Published 28 May 2014

Academic Editor: Ameur Cherif

Copyright © 2014 Faten Ghodhbane-Gtari et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reconciling the irreconcilable is a primary struggle in aerobic nitrogen-fixing bacteria. Although nitrogenase is oxygen and reactive oxygen species-labile, oxygen tension is required to sustain respiration. In the nitrogen-fixing Frankia, various strategies have been developed through evolution to control the respiration and nitrogen-fixation balance. Here, we assessed the effect of different oxygen tensions on Frankia sp. strain CcI3 growth, vesicle production, and gene expression under different oxygen tensions. Both biomass and vesicle production were correlated with elevated oxygen levels under both nitrogen-replete and nitrogen- deficient conditions. The mRNA levels for the nitrogenase structural genesnif ( HDK) were high under hypoxic and hyperoxic conditions compared to oxic conditions. The mRNA level for the hopanoid biosynthesis genes (sqhCandhpnC) was also elevated under hyperoxic conditions suggesting an increase in the vesicle envelope. Under nitrogen-deficient conditions, the hup2mRNA levels increased with hyperoxic environment, while hup1 mRNA levels remained relatively constant. Taken together, these results indicate that Frankia protects nitrogenase by the use of multiple mechanisms including the vesicle-hopanoid barrier and increased respiratory protection.

1. Introduction host plants and aerobically in culture [9–15]. The oxygen- labile nitrogenase enzyme is localized within specialized The genus Frankia is comprised of nitrogen-fixing actinobac- thick-walled structures, termed vesicles that are formed in teria that are able to establish a mutualistic symbiosis with planta and in vitro [2, 16–18]. Their shape is strain depen- a variety of dicotyledonous host plants that results in the dent and host-plant-influenced. Vesicles act as specialized establishment of a root nodule structure [1–6]. The bacteria structures for the nitrogen fixation process and are formed nourish their host plant with combined nitrogen and the terminally on short side branches of hyphae that have a plantsprovideinreturncarbonandenergy.Thissymbiosis septum near their base. The mature vesicle is surrounded allows actinorhizal host plants to colonize nutrient-poor by an envelope that extends down the stalk of the vesicle soils. Besides its life style within the host plant, these bacteria past the basal septum, which separates the vesicle from the are members of soil community although less information hypha. The envelope surrounding the vesicle is composed is known about this life style [7]. Under arid tropic and of multilaminated lipid layers containing primarily bacterio- subtropic conditions of North Africa, actinorhizal plants are hopanetetrol and its derivatives [19–22]. It is believed that this essentially represented by fast growing and highly tolerant lipidenvelopeactsasanoxygendiffusionbarriertoprotect trees from the family Casuarinaceae [8]. the nitrogenase enzyme from oxygen inactivation [19]. Under atmospheric oxygen conditions, Frankia actively Unlike other actinorhizal plants, Frankia found within fixes dinitrogen to ammonium within the root nodules of the the root nodules of Casuarina and Allocasuarina plants are 2 BioMed Research International devoid of symbiotic vesicle structures [23, 24]. A positive 2.3. Determination of Ammonia. Ammonium concentration correlation was observed between the differentiation of intra- was determined in cell-free media using modified protocol of cellular hyphae and the lignifications of the host-infected cell Berthelot’s reagent [47]. walls [23]. In several actinorhizal nodules, a low oxygen tension was shown to be consistent with the high concentrations 2.4. RNA Extraction, RT-PCRs, and Q-PCR. For these exper- of hemoglobin [2]. Frankia are known to produce truncated iments, all solutions and materials were DEPC-treated to hemoglobins [25–27]. Besides hemoglobins, Frankia possess prevent RNA degradation. RNA extractions were performed hydrogenases that may act as oxygen-scavenging enzymes by the Triton X100 method as previously described [48]. RNA [28]. Sequencing of several Frankia genomes [29–34]has samples were treated with DNase I (New England Biolabs) provided insight on the physiology and opened up new according to the manufacturer’s recommendations. RNA genomics tools for these microbes. These databases have samples were quantified with a Nanodrop 2000c spectropho- − ∘ been used in transcriptomics [35–37]andproteomicsstudies tometer (Thermo Scientific) and stored at 80 Cuntiluse. [38–40]onthesebacteria.Theaimofthepresentstudy The cDNA synthesis was performed using hexamer primers, was to investigate the expression levels for several selected 400 ng RNA and SuperScript III reverse transcriptase (Invit- genes involved under different oxygen concentration for the rogen) according to the manufacturer’s recommendations. Casuarina compatible Frankia sp. strain CcI3. These genes The cDNA was quantified by a Nanodrop 2000c spectropho- 𝜇 were involved in the following functions: nitrogen fixation tometer, diluted to 10 ng/ L working stocks in DNAse-free, − ∘ and assimilation, hopanoid biosynthesis, hydrogen uptake, RNAse-free H2O, and stored at 20 Cuntiluse. and oxidative stress. Frankia gene expression analyses were performed by qRT-PCR using specific primersTable ( 1)andSYBRGreen 2. Materials and Methods PCR Master Mix (Applied Biosystems) as described previously [49]. Briefly, each 25 𝜇L reaction contained 50 ng tem- 2.1. Culture Conditions and Experimental Design. Frankia sp. plate cDNA, 300 nM of the forward and reverse primer mix, ∘ and SYBR Green PCR Master Mix. Parameters for the Agilent strain CcI3 [41] was grown and maintained at 28 Cinbasal ∘ MP3000 were as follows: (1) 95 C for 15 min, (2) 40 cycles of MP growth medium with 5.0 mM propionate and 5.0 mM ∘ ∘ 95 Cfor15sand60C for 30 s, and (3) thermal disassociation NH4Cl as carbon and nitrogen sources, respectively, as ∘ ∘ cycle of 95 Cfor60s,55 C for 30 s, and incremental increases described previously [42]. ∘ In all experimental procedures, Frankia cells were grown in temperature to 95 C for 30 s. Reactions were performed for 7 days in 250 mL cylindrical bottles with a working in triplicates and the comparative threshold-cycle method was used to quantify gene expression. The results were MP medium volume of 50 mL with and without NH4Cl for nitrogen-deficient and nitrogen-replete conditions, respec- standardized with rpsA expression levels. Relative expression tively. Three sets of oxygen tensions were considered: oxic (fold changes) was determined by the Pfaffl method [50]with the control as the calibrator. Two biological replicates of the (atmospheric condition), hypoxic (reduced partial pressure triplicate samples were averaged. of oxygen), and hyperoxic (elevated oxygen levels). Hypoxic conditions were generated by placing the cultures in Brewer’s 3. Results jar that contained reduced partial pressures of oxygen by the use of gas packets (BBL GasPak BBL CampyPak System). 3.1. Growth and Vesicle Production under Different Oxygen For this system, water interacts with catalyst in the packet Pressures. Figure 1 shows the effect of oxygen on the growth generating a reduced partial pressure of oxygen within the yield of Frankia sp. strain CcI3. Under nitrogen-replete chamber. Hyperoxic conditions were generated by continu- conditions (NH4), the biomass of cells grown under hyper- ously air-sparging the cultures via an aquarium pump. oxic conditions was greater than both cultures grown under oxic and hypoxic conditions. Under nitrogen-deficient (N2) 2.2. Growth Assessment and Vesicle Count. For dry weight conditions, the biomass correlated with the oxygen level with determinations, cell cultures were collected on tarred mem- the hyperoxic conditions generating the greatest biomass. brane filters (type HA, 0.45 um pore size; Millipore Corp.). Furthermore, vesicle production under nitrogen-deficient The filters were placed in a Petri dish over desiccant and (N2) conditions positively correlated with oxygen tension. ∘ Cells under hyperoxic (air-sparged) conditions produced 2.6- dried at 90 Ctoconstantweight[43]. In parallel, protein 6 content was measured. Briefly, cell samples were solubilized and 5.4-fold more vesicles (6.50 ± 0.41 × 10 /mg) than oxic ∘ 6 6 by heating for 15 min at 90 Cin1.0NNaOHandtotalproteins (2.45 ± 0.29 × 10 /mg) and hypoxic (1.20 ± 0.36 × 10 /mg) were measured using BCA method [44]. conditions, respectively. Analysis of ammonia metabolism by Vesicle numbers were determined as previously described Frankia CcI3 indicates that it was correlated with oxygen [45, 46]. Briefly, cells were sonicated for 30 s with a Braun tension. With nitrogen-replete conditions, hyperoxic condi- model 350 sonifier under power setting of 3 using microtip tions resulted in the highest ammonia consumption, followed probe. This treatment disrupted the mycelia and released by oxic condition and lastly hypoxic condition (Figure 1(c)). vesicles. The numbers of vesicles were counted by using Under nitrogen-deficient conditions the level of ammo- a Petroff-Hausser counting chamber with a phase-contrast nium ions increased under lower oxygen tension. This level microscope at magnification of 400x. decreasedwithcorrespondingincreasesinoxygentension. BioMed Research International 3

Table 1: Primers used in this study.

Locus tag Gene Gene identity Sequence 󸀠 󸀠 5 -CGACAACGACATGAAGACC-3 francci3 4488 nif H Nitrogenase reductase iron-sulfur protein 󸀠 󸀠 5 -CTTGCCGATGATGCTCTC-3 󸀠 󸀠 5 -AAGGACATCGTCAACATCAGCCAC-3 francci3 4487 nif D Nitrogenase molybdenum-iron protein alpha chain 󸀠 󸀠 5 -AACTGCATCGCGGCGAAGTTATTC-3 󸀠 󸀠 5 -TGACGACGACTCCGGAAACAAACA-3 francci3 4486 nif K Nitrogenase molybdenum-iron protein beta chain 󸀠 󸀠 5 -TGTGGTAGACCTCGTCCTTGAACA-3 󸀠 󸀠 5 -AACAAATCTGCGACGTCACGGTCA-3 francci3 4496 hup1 Nickel-dependant hydrogenase, large subunit 󸀠 󸀠 5 -ACTCTCGATCCATTCACCGCAGTA-3 󸀠 󸀠 5 -TGGAAGGTCAACTGGCTGGAGAA-3 francci3 1076 hup2 Uptake hydrogenase, large subunit 󸀠 󸀠 5 -ATGTCTAGGCAGTACCGGAGGAAGAA-3 󸀠 󸀠 5 -GGGACGCCTGGCTGAAGA-3 francci3 1149 hboO Truncated hemoglobin 󸀠 󸀠 5 -CCAGAGCTGCCTGTCGAGATC-3 󸀠 󸀠 5 -CACCCCTCTTTGCCAACCG-3 francci3 2581 hboN Truncated hemoglobin 󸀠 󸀠 5 -GGTGGTTTCCGTCGGGAC-3 󸀠 󸀠 5 -TGCAATGGCTGCTGGACAA-3 francci3 0823 sqhC Squalene hopene cyclase 󸀠 󸀠 5 -TGCCGTAGACGTGGTTGAT-3 󸀠 󸀠 5 -AACTTCCCGGTCTCGCCGTT-3 francci3 0819 hpnC Squalene synthase 󸀠 󸀠 5 -AACGCGTTGAAGTGGAAACGAACC-3 󸀠 󸀠 5 -ACATGCCGGTGTTCTTCATTCAGG-3 francci3 2949 katA Catalase 󸀠 󸀠 5 -ACATCATCATGTGGCATCGACTCGG-3 󸀠 󸀠 5 -GTGCCAATGACACCCTTGAGAAGA-3 francci3 2817 sodA Superoxide dismutase 󸀠 󸀠 5 -AGTGGAGAATATGCCCGGAAAGGT-3 󸀠 󸀠 5 -TGCATGCGACGAACAACTTCCC-3 francci3 3012 gltD Glutamate synthase, small subunit 󸀠 󸀠 5 -ATGATGCTGACCTCGATCTGCTTG-3 󸀠 󸀠 5 -CGTGCTGAAGGTGATGTCCAAGAT-3 francci3 3013 gltB Glutamate synthase, large subunit 󸀠 󸀠 5 -AAATAGGCGTCGATCAGTTCCTGG-3 󸀠 󸀠 5 -ATGACCCGATCACCAAGGAACAGT-3 francci3 3142 glnA Glutamine synthetase, type I 󸀠 󸀠 5 -GGGTTGTAGTCATAACGGACATCG-3 󸀠 󸀠 5 -AACTTCTCCACCAGGCAGACGAT-3 francci3 3143 glnA Glutamine synthetase, type II 󸀠 󸀠 5 -AGAACTTGTTCCACGGAGCTGTCT-3 󸀠 󸀠 5 -TACAACATCGACTACGCGCTTTCC-3 francci3 4059 glnA Glutamine synthetase, catalytic region 󸀠 󸀠 5 -ATACCGGAACACGATCTCGAACTG-3 󸀠 󸀠 5 -CGAAGTCCGTTCCGAGTTC-3 francci3 1057 rpsA 30S ribosomal protein S1 󸀠 󸀠 5 -CGCCGAAGTTGACGATGG-3 Locus tag and gene designation were determined from the Integrated Microbial Genomes System (IMG) at the Joint Genome Institute (https://img.jgi.doe.gov/) [51].

3.2. Expression of Nitrogen Fixation and Assimilation Genes glutamine synthetase orthologs found within the Frankia sp. under Different Oxygen Pressures. The effect of oxygen on the strain CcI3 genome. We were able to follow the expression expression of several genes involved in nitrogen fixation and of three of these glnA genes (Figure 2(c)). The level of assimilation was measured by detecting changes in mRNA francci3 3143 mRNA was controlled by nitrogen. Under all levels via qRT-PCR (Figure 2). For nitrogen-deficient con- oxygen conditions, francci3 3143 mRNA levels increased 10– ditions, the level of structural nitrogenase genes (nif HDK) 15-fold under nitrogen-deficient (N2) conditions. Both high mRNA increased >10-fold under hyperoxic and hypoxic and low oxygen tensions increased the level of francci3 3143 conditions compared to oxic condition (Figure 2(a)). Under mRNA. The level of francci3 3142 mRNA was decreased nitrogen-replete conditions, the expression levels for these under nitrogen-deficient (N2) conditions and showed 7-fold genes were very low and there was no change with different increase under hyperoxic under nitrogen-replete conditions. oxygen tensions. The levels of francci3 4059 mRNA remained constant except The Frankia genome contains two glutamate synthase under hyperoxic conditions, in which levels increased 15- genes (gltBandgltD) encoding the large and small subunits of fold. Under hyperoxic conditions, the levels of francci3 4059 the enzyme. These two glutamate synthase genes were studied mRNA were controlled by nitrogen status and increased for their expression levels under three oxygen tensions. The approximately 2-3-fold from nitrogen-replete (NH4)condi- mRNA levels of the gltB gene were reduced except under tions. hyperoxic and nitrogen-replete conditions (Figure 2(b)). The gltD mRNA levels increased slightly (1.3–2.5-fold) under the 3.3. Expression of Genes Known to Protect Nitrogenase from different nitrogen and oxygen conditions. There were four Oxygen and Reactive Oxygen Species. The biosynthesis of 4 BioMed Research International

0.3 0.16 0.14 0.25 0.12 0.2 0.1 0.15 0.08 0.06 0.1 Protein (mg/mL) Protein

Dry (mg/mL) weight 0.04 0.05 0.02 0 0 LN2 LNH4 NN2 NNH4 HN2 HNH4 LN2 LNH4 NN2 NNH4 HN2 HNH4

(a) (b) 70

Ammonium ions (mg/L) ions Ammonium 10

0 LN2 LNH4 NN2 NNH4 HN2 HNH4

(c)

Figure 1: Biomass yields of Frankia sp. strain CcI3 grown under nitrogen fixation (N2) and nitrogen-replete (NH4) at hypoxic (L), oxic (N), and hyperoxic (H) conditions as estimation by (a) dry weight and (b) total protein and determination of (c) ammonium ion concentrations. hopanoids has been correlated with vesicle development the oxygen tension levels. Under hypoxic nitrogen-deficient [19]. The effect of oxygen tension on the expression of the conditions, mRNA levels for hboNincreasedabout1.5-fold. squalene synthase (hpnC) and squalene/phytoene cyclase The effects of oxygen tension and nitrogen status onthe (sqhC) genes was examined (Figure 2(d)). Under nitrogen- expression levels of two oxygen defense enzymes, catalase replete conditions (NH4), the level of mRNA for sqhCshowed (katA) and superoxide dismutase (sodA), were also tested a 2-fold increase for hyperoxic conditions. A smaller increase (Figure 2(g)). Under hyperoxic conditions, the mRNA levels was observed for hpnCmRNAlevels.Ingeneral,sqhCand of katA increased 6.5- and 8-fold under nitrogen-deficient hpnC were expressed constitutively with comparable mRNA (N2) and nitrogen-replete (NH4) conditions, respectively. The levels for hypoxic and oxic levels. Under nitrogen-deficient expression of the sodAgeneappearedtobeconstitutiveunder (N2) conditions, the mRNA levels of both genes (sqhCand all oxygen tensions and both nitrogen statuses. hpnC) increased 2- and 1.5-fold, respectively. The Frankia CcI3 genome contains two hydrogenase 4. Discussion operons [30, 52, 53]. We tested the effects of oxygen tension and nitrogen status of their gene expression levels Without a doubt, the vesicle is the most characteristic (Figure 2(e)). Under nitrogen-replete (NH4) conditions, the morphogenetic structure produced by Frankia [1]. Vesicles level of mRNA for hup2 increased proportionally with the are functionally analogous to cyanobacterial heterocysts level of oxygen present, while the level of mRNA for hup1 providing unique specialized cells that allow nitrogen fixation only increased under hyperoxic conditions. The expression of under aerobic condition [54, 55]. In this study, the growth hup2wasinfluencedbythenitrogenstatusofthecellsandby of Frankia strain CcI3 was evaluated under three oxygen the oxygen levels. Under both conditions, hup2mRNAlevels tensions. The results indicate that growth increased with increased, but hup1 expression remained constant. elevated oxygen tensions (Figure 1)confirmingtheaerobic The effect of oxygen tension and nitrogen status was nature of the microbe. Although the dry weight measurement investigated on the expression of two truncated hemoglobins increased, the total protein values were reduced under hyper- (hboOandhboN).ThelevelofmRNAofhboOandhboN oxic nitrogen-deficient (N2) conditions. This result would increased under hyperoxic condition for both nitrogen con- imply that the cells were producing other metabolic products ditions (Figure 2(f)). Under nitrogen-replete (NH4)condi- under this condition and a similar level of protein compared tions, mRNA levels for hboO increased proportionally to to hypoxic nitrogen-deficient (N2) condition. Thus, this result BioMed Research International 5

250 3 35 2.5 30 200 25 2 150 20 1.5 100 15 1 10 50 0.5 5 0 0 0 LN2 LNH4 NN2 NNH4 HN2 HNH4 LN2 LNH4 NN2 NNH4 HN2 HNH4 LN2 LNH4 NN2 NNH4 HN2 HNH4

nifH nifK gltD francci3 3142 francci3 4059 nifD gltB francci3 3143 (a) (b) (c) 6 30 5 4.5 5 25 4 4 20 3.5 3 3 15 2.5 2 2 10 1.5 1 5 1 0.5 0 0 0 LN2 LNH4 NN2 NNH4 HN2 HNH4 LN2 LNH4 NN2 NNH4 HN2 HNH4 LN2 LNH4 NN2 NNH4 HN2 HNH4

sqhC hup2 HboO hpnC hup1 HboN (d) (e) (f) 9 8 7 6 5 4 3 2 1 0 LN2 LNH4 NN2 NNH4 HN2 HNH4

SodA KatA (g)

Figure 2: Relative gene expression (fold change) in response to hyperoxic and hypoxic conditions. Frankia cultures were grown under nitrogen-replete (NH4) or nitrogen-deficient 2(N ) conditions. These cultures were exposed to oxic (N), hyperoxic (H), and hypoxic (L) conditions as described in Section 2. Experimental gene expression was normalized to the rpsA housekeeping gene and compared to the calibrator (NH4 oxic conditions). The following genes were analyzed: (a) nif HDK (b) gltBandgltD, (c) glnA genes, (d) hpnCandsqhC, (e) hup1andhup2, (f) hboNandhboO, and (g) sodAandkatA. suggests that part of the respiration was uncoupled providing tensions. The numbers of vesicles produced per mg dry some oxygen protection. Frankia contains two respiratory weight increased with elevated oxygen levels. These results systems and a cyanide-insensitive system was proposed to confirmthoseobtainedpreviously[58, 59]. help protect nitrogenase from oxygen inactivation [46]. With In our study, we investigated the effects of oxygen on other aerobic nitrogen-fixing bacteria, increased respiratory gene expression for a variety of functional genes involved in rates in response to elevated oxygen tensions help maintain nitrogen fixation, nitrogen assimilation, and protection from low levels of intracellular oxygen protecting nitrogenase from oxygen and other reactive oxygen species [60]. The levels inactivation [56, 57]. Under nitrogen-deficient (N2)condi- of expression for the structural nitrogenase genes (nif HDK) tions, vesicles were produced and correlated with oxygen indicate a concordant profile with clear induction under 6 BioMed Research International

nitrogen-deficient (N2) conditions. Transcriptome studies alni ACN14a [60]. We did not test anoxic conditions in our on Frankia sp. strain CcI3 under nitrogen-deficient and study. nitrogen-replete conditions also show an increase in nif HDK Increasedoxygentensioncanleadtoelevatedoxidative gene expression [35, 36]. The levels of nif HDK mRNA stress conditions. We investigated the influence of oxygen showed an increase under hypoxic and hyperoxic conditions tensions on reactive oxidative stress genes. While sodA indicating that nitrogenase induction was influenced by expression levels were constitutive, katA gene expression oxygen levels. increased under hyperoxic conditions. In general, our results The hopanoid envelope has been postulated tobe confirm those of Steele and Stowers [63], which examined enzymatic activity levels. They reported an increase in cata- involved in the protection of nitrogenase from oxygen lase activity in cultures derepressed for nitrogen fixation inactivation [19]. We found that mRNA levels of squalene compared to ammonium-grown cultures. synthase (hpnC) and squalene-hopene cyclase (sqhC) genes increased in response to oxygen tension under nitrogen- deficient conditions, but remained constant under nitrogen- Conflict of Interests replete conditions (Figure 2(d)). The results correlate with The authors declare that there is no conflict of interests the increase in vesicle envelope observed under high oxygen regarding the publication of this paper. levels [61]. Nalin et al. [62]foundonlyaslightlyhigher hopanoid content under nitrogen-deficient conditions suggesting remobilization rather than nascent biosynthesis. Fur- Acknowledgments thermore, the Frankia sp. strain CcI3 transcriptome profiles LouisS.TisawassupportedinpartbyAgricultureandFood under nitrogen-deficient and nitrogen-replete conditions did Research Initiative Grant 2010-65108-20581 from the USDA not show any significant differences in hopanoid biosynthetic NationalInstituteofFoodandAgriculture,HatchGrant genes [35, 36]. However, these studies were performed under NH530, and the College of Life Sciences and Agriculture one oxygen tension while our study has investigated three at the University of New Hampshire, Durham, NH, USA. different oxygen tensions. This is scientific contribution number 2556 from the NH Analysisofthenitrogenassimilationgenes(gltB, gltD, Agricultural Experimental Station. Maher Gtari and Faten and glnA) is a bit more complex. The Frankia CcI3 genome Ghodhbane-Gtari were supported in part by a Visiting contained several homologues of glnA. The mRNA level of Scientist and Postdoctoral Scientist Program administered by francci3 3143 correlated the best with nitrogen regulation, the NH AES at the University of New Hampshire. being increased under nitrogen-deficient conditions. Tran- scriptome studies have shown that francci3 3143 expression increased significantly under nitrogen-fixing conditions35 [ , References 36], while all of the other homologues remained consistent. [1]D.R.BensonandW.B.Silvester,“BiologyofFrankia strains, This result would suggest that this gene encoded primary actinomycete symbionts of actinorhizal plants,” Microbiological nitrogen scavenging enzyme. The levels of expression were Reviews,vol.57,no.2,pp.293–319,1993. also influenced by elevated oxygen tensions during increased [2] K. Huss-Danell, “Actinorhizal symbioses and their N2 fixation,” nitrogenase activity. The expression levels of the gltBandgltD New Phytologist,vol.136,no.3,pp.375–405,1997. appear to be less influenced by oxygen tension. These effects [3] L. G. Wall, “The actinorhizal symbiosis,” Journal of Plant Growth seemed in agreement with the ammonia metabolism results Regulation,vol.19,no.2,pp.167–182,2000. that showed an increase in consumption under hyperoxic conditions. [4] C. Santi, D. Bogusz, and C. Franche, “Biological nitrogen fixation in non-legume plants,” Annals of Botany, vol. 111, no. 5, pp. Our results on hemoglobin gene expression correlate with 743–767, 2013. previous results [48]thatshowednoincreaseinhboNand [5] J.SchwenckeandM.Caru,´ “Advances in actinorhizal symbiosis: hboO expression in response to nitrogen status increased host plant-Frankia interactions, biology, and applications in under low oxygen tension. However, our results conflict in arid land reclamation. A review,” Arid Land Research and response to oxygen. We found that both hboNandhboO Management,vol.15,no.4,pp.285–327,2001. mRNA levels increased under hyperoxic conditions. The use [6] A. Sellstedt and K. H. Richau, “Aspects of nitrogen-fixing actin- of the more sensitive qRT-PCR in our study compared to RT- obacteria, in particular free-living and symbiotic Frankia,” PCR is the best explanation for these differences. FEMS Microbiology Letters,vol.342,no.2,pp.179–186,2013. Frankia possesses two uptake hydrogenase systems [52, [7]E.E.Chaia,L.G.Wall,andK.Huss-Danell,“Lifeinsoilbythe 53]. One of them has been correlated with symbiotic growth actinorhizal root nodule endophyte Frankia.Areview,”Symbi- and the other to free-living conditions [53]. Our results show osis, vol. 51, no. 3, pp. 201–226, 2010. that hup2 gene expression was influenced by nitrogen status [8] M. Gtari and J. O. Dawson, “An overview of actinorhizal plants suggesting that it was associated with vesicle production, in Africa,” Functional Plant Biology,vol.38,no.8-9,pp.653–661, while hup1 gene expression was relatively constant. The 2011. levels of hup2 mRNA increased proportionally with oxygen [9]J.D.Tjepkema,W.Ormerod,andJ.G.Torrey,“Vesiclefor- tensions suggesting potential oxygen protection mechanism. mation and acetylene reduction activity in Frankia sp. CPI1 Anoxic conditions have no effect on hydrogenase gene cultured in defined nutrient media,” Nature,vol.287,no.5783, expression by Frankia CcI3 but increased by 30% for Frankia pp. 633–635, 1980. BioMed Research International 7

[10] J. D. Tjepkema, W.Ormerod, and J. G. Torrey, “Factors affecting concentration, and nitric oxide,” Canadian Journal of Microbi- vesicle formation and acetylene reduction (nitrogenase activity) ology,vol.55,no.7,pp.867–873,2009. in Frankia sp. CpI1,” Canadian Journal of Microbiology,vol.27, [26] J. D. Tjepkema, “Hemoglobins in the nitrogen-fixing root no. 8, pp. 815–823, 1981. nodules of actinorhizal plants,” Canadian Journal of Botany,vol. [11] D. Gauthier, H. G. Diem, and Y. Dommergues, “In vitro nitro- 61, no. 11, pp. 2924–2929, 1983. gen fixation by 2 actinomycete strains isolated from Casuarina [27] J. D. Tjepkema, R. E. Cashon, J. Beckwith, and C. R. Schwintzer, nodules,” Applied and Environmental Microbiology,vol.41,pp. “Hemoglobin in Frankia, a nitrogen-fixing actinomycete,” 306–308, 1981. AppliedandEnvironmentalMicrobiology,vol.68,no.5,pp. [12] M. A. Murry, Z. Zhongze, and J. G. Torrey, “Effect of O2 on 2629–2631, 2002. vesicle formation, acetylene reduction, and O2-uptake kinetics [28] A. Sellstedt, P. Reddell, and P. Rosbrook, “The occurrence in Frankia sp. HFPCc13 isolated from Casuarina cunninghami- of hemoglobin and hydrogenase in nodules of 12 Casuarina- ana,” Canadian Journal of Microbiology,vol.31,no.9,pp.804– Frankia symbiotic associations,” Physiologia Plantarum,vol.82, 809, 1985. no.3,pp.458–464,1991. [13]Z.ZhongzeandJ.G.Torrey,“Biologicalandculturalcharacter- [29] F. Ghodbhane-Gtari, N. Beauchemin, D. Bruce et al., “Draft istics of the effective Frankia strain HFPCcl3 (Actinomycetales) genome sequence of Frankia sp. strain CN3, an atypical, from Casuarina cunninghamiana (Casuarinaceae),” Annals of non-infective (Nod-) ineffective (Fix-) isolate from Coriaria Botany,vol.56,no.3,pp.367–378,1985. nepalensis,” Genome Announcements,vol.1,no.2,ArticleID [14]M.S.Fontaine,S.A.Lancelle,andJ.G.Torrey,“Initiationand e0008513, 2013. ontogeny of vesicles in cultured Frankia sp. strain HFPArI3,” [30]P.Normand,P.Lapierre,L.S.Tisaetal.,“Genomecharacter- Journal of Bacteriology,vol.160,no.3,pp.921–927,1984. istics of facultatively symbiotic Frankia sp. strains reflect host [15] A. J. P. Burggraaf and W. A. Shipton, “Studies on the growth of range and host plant biogeography,” Genome Research,vol.17, Frankia isolates in relation to infectivity and nitrogen fixation no.1,pp.7–15,2007. (acetylene reduction),” Canadian Journal of Botany,vol.61,no. [31] A. Sen, N. Beauchemin, D. Bruce et al., “Draft genome sequence 11, pp. 2774–2782, 1983. of Frankia sp. strain QA3, a nitrogen-fixing actinobacterium [16] T. M. Meesters, “Localization of nitrogenase in vesicles isolated from the root nodule of Alnus nitida,” Genome of Frankia sp. Cc1.17 by immunogoldlabelling on ultrathin Announcements, vol. 1, no. 2, Article ID e0010313, 2013. cryosections,” Archives of Microbiology,vol.146,no.4,pp.327– 331, 1987. [32] S. R. Mansour, R. Oshone, S. G. Hurst, K. Morris, W.K. Thomas, andL.S.Tisa,“DraftgenomesequenceofFrankia sp. strain [17]T.M.Meesters,S.T.VanGenesen,andA.D.L.Akkermans, CcI6, a salt-tolerant nitrogen-fixing actinobacterium isolated “Growth, acetylene reduction activity and localization of nitro- from the root nodule of Casuarina cunninghamiana,” Genome genase in relation to vesicle formation in Frankia strains Cc1.17 Announcements,vol.2,no.1,ArticleIDe01205-13,2014. and Cp1.2,” Archives of Microbiology,vol.143,no.2,pp.137–142, 1985. [33] I. Nouioui, N. Beauchemin, M. N. Cantor et al., “Draft genome sequence of Frankia sp.strainBMG5.12,anitrogen- [18]T.M.Meesters,W.M.Vanvliet,andA.D.L.Akkermans, fixing actinobacterium isolated from Tunisian soils,” Genome “Nitrogenase is restricted to the vesicles in Frankia strain Announcements,vol.1,no.4,ArticleIDe00468-13,2013. EAN1pec,” Physiologia Plantarum,vol.70,no.2,pp.267–271, 1987. [34] L. G. Wall, N. Beauchemin, M. N. Cantor et al., “Draft genome [19]A.M.Berry,O.T.Harriott,R.A.Moreau,S.F.Osman,D.R. sequence of Frankia sp. strain BCU110501, a nitrogen-fixing Benson, and A. D. Jones, “Hopanoid lipids compose the Frankia actinobacterium isolated from nodules of Discaria trinevis,” vesicle envelope, presumptive barrier of oxygen diffusion to Genome Announcements,vol.1,no.4,ArticleIDe00503-13, nitrogenase,” Proceedings of the National Academy of Sciences of 2013. the United States of America,vol.90,no.13,pp.6091–6094,1993. [35] D. M. Bickhart and D. R. Benson, “Transcriptomes of Frankia [20] A. M. Berry, R. A. Moreaua, and A. D. Jones, “Bacterio- sp. strain CcI3 in growth transitions,” BMC Microbiology,vol.11, hopanetetrol: abundant lipid in Frankia cells and in nitrogen- article 192, 2011. fixing nodule tissue,” Plant Physiology, vol. 95, no. 1, pp. 111–115, [36] H.-I. Lee, A. J. Donati, D. Hahn, L. S. Tisa, and W.-S. 1991. Chang, “Alteration of the exopolysaccharide production and [21] O. T. Harriott, L. Khairallah, and D. R. Benson, “Isolation and the transcriptional profile of free-living Frankia strain CcI3 structure of the lipid envelopes from the nitrogen-fixing vesicles under nitrogen-fixing conditions,” Applied Microbiology and of Frankia sp. strain CpI1,” Journal of Bacteriology,vol.173,no. Biotechnology,vol.97,no.24,pp.10499–10509,2013. 6, pp. 2061–2067, 1991. [37] N. Alloisio, C. Queiroux, P. Fournier et al., “The Frankia [22]H.C.Lamont,W.B.Silvester,andJ.G.Torrey,“Nileredfluo- alni symbiotic transcriptome,” Molecular Plant-Microbe Interac- rescence demonstrates lipid in the envelope of vesicles from N2- tions,vol.23,no.5,pp.593–607,2010. fixing cultures of Frankia,” Canadian Journal of Microbiology, [38] N. Alloisio, S. Felix,J.Mar´ echal´ et al., “Frankia alni proteome vol. 34, no. 5, pp. 656–660, 1988. under nitrogen-fixing and nitrogen-replete conditions,” Physi- [23] R. H. Berg and L. McDowell, “Endophyte differentiation in ologia Plantarum,vol.130,no.3,pp.440–453,2007. Casuarina actinorhizae,” Protoplasma,vol.136,no.2-3,pp.104– [39] J. E. Mastronunzio and D. R. Benson, “Wild nodules can be 117, 1987. broken: proteomics of Frankia in field-collected root nodules,” [24] R. H. Berg and L. Mcdowell, “Cytochemistry of the wall of Symbiosis,vol.50,no.1-2,pp.13–26,2010. infected-cells in Casuarina actinorhizae,” Canadian Journal of [40] J. E. Mastronunzio, Y. Huang, and D. R. Benson, “Diminished Botany, vol. 66, no. 10, pp. 2038–2047, 1988. exoproteome of Frankia spp. in culture and symbiosis,” Applied [25] V. Coats, C. R. Schwintzer, and J. D. Tjepkema, “Truncated and Environmental Microbiology,vol.75,no.21,pp.6721–6728, hemoglobins in Frankia CcI3:effectsofnitrogensource,oxygen 2009. 8 BioMed Research International

[41] Z. Zhang, M. F. Lopez, and J. G. Torrey, “A comparison of Frankia sp. HFPCcI3 isolated from Casuarina cunninghamiana,” cultural characteristics and infectivity of Frankia isolates from Canadian Journal of Microbiology,vol.31,no.9,pp.804–809, root nodules of Casuarina species,” Plant and Soil,vol.78,no. 1985. 1-2, pp. 79–90, 1984. [59] Z. Zhongze, M. A. Murry, and J. G. Torrey, “Culture condi- [42] N. J. Beauchemin, T. Furnholm, J. Lavenus et al., “Casuarina tions influencing growth and nitrogen fixation in Frankia sp. root exudates alter the physiology, surface properties, and plant HFPCcI3 isolated from Casuarina,” Plant and Soil,vol.91,no.1, infectivity of Frankia sp. strain CcI3,” Applied and Environmen- pp. 3–15, 1986. tal Microbiology,vol.78,no.2,pp.575–580,2012. [60]K.H.Richau,R.L.Kudahettige,P.Pujic,N.P.Kudahettige, [43] L. Tisa, M. Mcbride, and J. C. Ensign, “Studies of growth andA.Sellstedt,“Structuralandgeneexpressionanalysesof and morphology of Frankiastrains EAN1pec, EuI1c, CpI1, and uptake hydrogenases and other proteins involved in nitrogenase ACN1AG,” Canadian Journal of Botany, vol. 61, no. 11, pp. 2768– protection in Frankia,” JournalofBiosciences,vol.38,no.4,pp. 2773, 1983. 703–712, 2013. [44] P. K. Smith, R. I. Krohn, G. T. Hermanson et al., “Measurement [61] R. Parsons, W. B. Silvester, S. Harris, W. T. Gruijters, and of protein using bicinchoninic acid,” Analytical Biochemistry, S. Bullivant, “Frankia vesicles provide inducible and absolute vol. 150, no. 1, pp. 76–85, 1985. oxygen protection for nitrogenase,” Plant Physiology,vol.83,no. [45] L. S. Tisa and J. C. Ensign, “Comparative physiology of nitroge- 4, pp. 728–731, 1987. nase activity and vesicle development for Frankia strains CpI1, [62] R. Nalin, S. R. Putra, A.-M. Domenach, M. Rohmer, F. Gour- AG ACN1 ,EAN1pec and EUN1f,” Archives of Microbiology,vol.147, biere,andA.M.Berry,“Highhopanoid/totallipidsratioin no. 4, pp. 383–388, 1987. Frankia mycelia is not related to the nitrogen status,” Microbiol- [46] L. S. Tisa and J. C. Ensign, “The calcium requirement for ogy,vol.146,no.11,pp.3013–3019,2000. functional vesicle development and nitrogen fixation by Frankia [63] D. B. Steele and M. D. Stowers, “Superoxide dismutase and strains EAN1pec and CpI1,” Archives of Microbiology,vol.149,no. catalase in Frankia,” Canadian Journal of Microbiology,vol.32, 1,pp.24–29,1987. no.5,pp.409–413,1986. [47] E. D. Rhine, G. K. Sims, R. L. Mulvaney, and E. J. Pratt, “Improving the Berthelot reaction for determining ammonium in soil extracts and water,” Soil Science Society of America Journal, vol. 62, no. 2, pp. 473–480, 1998. [48] J. Niemann and L. S. Tisa, “Nitric oxide and oxygen regulate truncated hemoglobin gene expression in Frankia strain CcI3,” Journal of Bacteriology,vol.190,no.23,pp.7864–7867,2008. [49] F. Perrine-Walker, P. Doumas, M. Lucas et al., “Auxin carriers localization drives auxin accumulation in plant cells infected by Frankia in Casuarina glauca actinorhizal nodules,” Plant Physiology,vol.154,no.3,pp.1372–1380,2010. [50] M. W. Pfaffl, “A new mathematical model for relative quantification in real-time RT-PCR,” Nucleic Acids Research,vol.29,no. 9, article e45, 2001. [51] V. M. Markowitz, F. Korzeniewski, K. Palaniappan et al., “The integrated microbial genomes (IMG) system,” Nucleic Acids Research, vol. 34, pp. D344–D348, 2006. [52] P. Normand, C. Queiroux, L. S. Tisa et al., “Exploring the genomes of Frankia,” Physiologia Plantarum,vol.130,no.3,pp. 331–343, 2007. [53] M. Leul, P. Normand, and A. Sellstedt, “The organization, regulation and phylogeny of uptake hydrogenase genes in Frankia,” Physiologia Plantarum,vol.130,no.3,pp.464–470, 2007. [54] N. A. Noridge and D. R. Benson, “Isolation and nitrogen- fixing activity of Frankia sp. strain CpI1 vesicles,” Journal of Bacteriology,vol.166,no.1,pp.301–305,1986. [55] L. S. Tisa and J. C. Ensign, “Isolation and nitrogenase activity of vesicles from Frankia sp. strain EAN1pec,” Journal of Bacteriol- ogy,vol.169,no.11,pp.5054–5059,1987. [56] A. Hochman and R. H. Burris, “Effect of oxygen on acetylene reduction by photosynthetic bacteria,” Journal of Bacteriology, vol. 147, no. 2, pp. 492–499, 1981. [57] H. Dalton and J. R. Postgate, “Effect of oxygen on growth of Azotobacter chroococcum in batch and continuous cultures,” Journal of General Microbiology,vol.54,no.3,pp.463–473,1968. [58] M. A. Murry, Z. Zhongze, and J. G. Torrey, “Effect of O2 on vesicle formation, acetylene reduction, and O2-uptake kinetics in Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 317524, 11 pages http://dx.doi.org/10.1155/2014/317524

Research Article Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

Okba Selama,1 GregoryC.A.Amos,2 Zahia Djenane,1 Chiara Borsetto,2 Rabah Forar Laidi,3 David Porter,2 Farida Nateche,1 ElizabethM.H.Wellington,2 and Hocine Hacène1

1 Microbiology Group, Laboratory of Cellular and Molecular Biology, Faculty of Biological Sciences, USTHB, BP 32, EL ALIA, BabEzzouar,Algiers,Algeria 2 School of Life Sciences, University of Warwick, Coventry CV4 7AL, UK 3 Department de Biologie, Ecole Normale Superieure (ENS), Vieux Kouba, Alger, Algeria

Correspondence should be addressed to Hocine Hacene;` h [email protected]

Received 26 February 2014; Revised 13 April 2014; Accepted 6 May 2014; Published 27 May 2014

Academic Editor: Ameur Cherif

Copyright © 2014 Okba Selama et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

1. Introduction another niche harbouring “extremophiles” [8]; major catego- ries of extremophiles include halophiles, thermophiles, aci- There is an increasingly urgent need for new active biomole- dophiles, alkaliphiles, and haloalkaliphiles [6, 9]. cules and enzymes for use in industry and therapy [1]. The haloalkaliphiles bacteria have attracted a great deal of However, the rate of discovery of new useful compounds has attention from researchers in this last decade [9]. In 1982, the been in decline [2, 3] and because of this there is an interest in term haloalkaliphile was used for the first time to describe investigating previously unexplored ecological niches [4, 5], bacteria that are both halophilic and alkaliphilic [10]. This particularly extreme environments. These environments have group of bacteria is able to grow optimally or very well at pH provided a useful source of novel biologically active com- values at or above 10 along with high salinity (up to 25% (w/v) pounds in recent years [1, 6, 7]. NaCl) [11]. Extreme environments are distributed worldwide. These To encounter such harsh conditions, haloalkaliphilic ecosystems were thought to be lifeless as insurmountable microorganisms have found various physiological strategies extreme physical and chemical barriers to life exhibit. With to sustain their cell structure and function [12, 13]. These the advancement of our knowledge, we now see them as yet bacteria have widely been identified and studied from the 2 BioMed Research International hypersaline environments, soda lakes, solar saltern, salt Spain brines, carbonate springs, and Dead Sea [14]. Their survival Mediterranean Sea obviously indicated the widespread distribution of such organisms in natural saline environments [12, 15]. Atlantic The interest in haloalkaliphilic microorganisms is due not Ocean only to the necessity for understanding the mechanisms of adaptation to multiple stresses and detecting their diversity, Biskra Morocco Djelfa Tunisia but also to their possible application in biotechnology [9]. The present work involved the isolation and characterization of new haloalkalitolerant and haloalkaliphilic bac- Bechar teria able to produce extremozymes and elaborate natural Ouargla bioactive compounds effective against pathogenic bacteria Algerian Sahara Libya and fungi as well. The screening for genes coding for putative antitumor compounds by PCR with three sets of primers Adrar In Salah wasalsoperformed.Wehavebeeninterestedinsoilsof Algerian Sahara Desert, which is one of the biggest deserts and encompasses one of the most extreme environments worldwide (Sabkha and Chott). However, it is also considered to be one of the less explored parts. Our team has been N Tamanrasset

interested in these magnificent ecosystems for many years Mauritania and the few studies that have been published have shown Niger great active biomolecules [16–18], biodiversity of interesting 300 Mali new taxa [19–24], and enzymes [25, 26]. (km) Figure 1: Location of the sampled sites from Algerian Sahara Desert. ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 Djelfa: 35 16 47.5 N343 25.4 E; Biskra: 34 11 01.1 N607 21.8 E; ∘ 󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 2. Material and Methods Ouargla: 33 29 28.85 N 5 59 10.52 E; Tamanrasset 23 00 30.01 N ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 5 13 33.32 E; In Salah: 27 11 31.60 N227 12.52 E; Adrar: ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 ∘ 󸀠 󸀠󸀠 2.1. Sampling and Strains Isolation. Samples from different 27 44 48.14 N016 10.21 W; Bechar: 30 51 25.71 N159 58.56 soils(7sites)ofAlgeria’sSaharaDesertwerecollectedon W. March2010(100–300gpersiteinsterilebags)(Figure 1). Most samples were saline and alkaline soils, with an electrical ∘ conductivity between 1.4 and 20.2 mS/cm (at 20 C) and pH 2.3. Molecular Study ∘ range of 7.5–9; the temperature varies from 22 Cnorthto ∘ 44 C south of the Sahara. One gram from each sample was 2.3.1. DNA Extraction. Total genomic DNA from the differ- suspended in 9 ml sterile water (of 0.9%, 10%, and 20% NaCl ent selected bacteria for this study was isolated and purified −4 w/v) and serial dilutions to 10 . For each dilution and for using Qiagen Blood and Tissue DNA extraction kit (Qiagen, each concentration, soil particles were allowed to sediment; UK). DNA was eluted in Tris-HCL and its quantity and quality were tested using NanoDrop 2000 (Thermo Scientific). then 0.1 mL of the liquid phase was spread onto the surface ∘ of each of the modified International Streptomyces Project 2 DNA was stored at −20 Cuntiluse. (ISP2) [27] media agar supplemented with NaCl with respect to the various concentrations of salt used for dilutions (0.9%, 2.3.2. Molecular Identification. The amplification of 16S 10%, and 20% NaCl w/v) and adjusted to either pH= 7 or rRNA gene for the selected strains was performed using pH=10byadding5MNaOHbeforeautoclavingandspread the universal bacterial primer pairs pA/pH designed by onto nutrient agar plates. The plates were maintained at Edwards et al. [28](Table 1). PCR reactions were performed ∘ ∘ constanthumidityincubatedateither30Cor50Cfor15 in final reaction volume of 50 𝜇L containing 1 𝜇L(10–100ng) days. Colonies were picked out and repeatedly restreaked of DNA template, 25 𝜇L master mix (Promega, Madison, until purity was confirmed. All bacterial culture isolates were WI, USA), 1 𝜇L(10𝜇M) of each primer, and 1 𝜇LofBSA ∘ stored at 4 C in the same medium used for isolation. (10 mg/mL) (Promega, Madison, WI, USA). PCR products were analyzed on 1.5% (w/v) agarose (Sigma, UK) in 40 mM Tris-acetate with 1 mM EDTA (TAE) buffer at pH of 8.0 −1 2.2. Physiological Growth Parameters. Physiological growth stained with ethidium bromide at 0.5 𝜇gmL .Bandsof parameters for the thirteen selected strains were determined the corresponding size were cut out and purified with by agar plate method on modified ISP2 medium depending gel extraction kit (Qiagen; Venlo, Netherlands) as per the on the modified parameter. Salinity tolerance was examined manufacturer’s instructions. for0,1,5,10,15,20,and25%NaClw/v.ThepHgrowthrange The nucleotide sequences for the 16S rRNA gene of the was investigated between pH 5 and 12 at intervals of 1 pH different strains were carried out by GATC Biotech (UK). unit. The temperatures tested were 4, 10, 15, 20, 25, 30, 37, The isolates were identified using the EzTaxon-e server ∘ 40, 42, 45, 55, and 60 C. Incubation time was one week for (http://eztaxon-e.ezbiocloud.net/)onthebasisof16SrRNA Actinobacteria and two days for non-Actinobacteria. sequence data [32]. BioMed Research International 3

Table 1: Primers used in this study.

Primers Gene Molecules Reference PCR programs PCR cycles were as follows: 1 cycle at ∘ ∘ pA: AGAGTTTGATCCTGGCTCAG 95 C for 10 min; 35 cycles at 94 Cfor ∘ ∘ pH: AAGGAGGTGATCCAGCCGCA 16S RNA /////////// [28] 1 min, 55 C for 1 min, and 72 Cfor ∘ 1,5 Kb 2 min; one final cycle at 72 Cfor 10 min. ∘ PCR conditions used were 95 Cfor Glu1: CSGGSGSSGCSGGSTTCATSGG ∘ ∘ dNDP-Glucose-4,6- 4 min; 30 cycles of 95 Cfor30s,65C Glu2: GGGWRCTGGYRSGGSCCGTAGTTG ////// [29] ∘ dehydratases for 30 s, and 68 C for 1.30 min; and a 546 bp ∘ final extension cycle at 68 C for 5 min. ∘ PCR protocol: 1 cycle of 95 Cfor Oxytryptophan ∘ ∘ 5 min; 7 cycles of 95 Cfor30sec,65C dimerization genes ∘ StaDVF: GTSATGMTSCAGTACCTSTACGC BE-54017, for 30 sec with 1 Cdecrementpercycle (StaD/RebD/VioB) ∘ ∘ StaDVR: YTCVAGCTGRTAGYCSGGRTG. (tryptophan [30] to 59 C, and 72 C for 40 sec; 30 cycles (indolotryptoline ∘ ∘ 570 bp dimmers) of 95 Cfor30sec,58Cfor30sec,and biosynthetic gene ∘ ∘ 72 C for 40 sec; 1 cycle of 72 Cfor cluster) ∘ 7 min; hold at 4 C Optimized PCR conditions were as ∘ Angucycline follows: (1) denaturation at 94 Cfor Iadomycin AuF3: GAACTGGCCSCGSRTBTT cyclases 5 min, (2) 30 amplification cycles with cyclase gene of ∘ AuR4: CCNGTGTGSARSKTCATSA [31] denaturation (45 s, 94 C), annealing Streptomyces ∘ 600–700 bp Marine (60 s, 60 C), and extension (60 s, venezuelae ISP5230 ∘ ∘ sponge 72 C), and (3) a final extension at 72 C for 8 min.

The Molecular Evolutionary Genetics Analysis (MEGA) The PCR mixture included 1-2 𝜇LofgenomicDNA, software, version 4.0.2, was used to assist the phylogenetic 15 𝜇L master mix (Sigma,UK), 1 𝜇L each of forward and analyses and the phylogenetic tree construction [33]. Similar reverse primers (10 𝜇M each) (Sigma, UK), 1 𝜇LofBSA 16S rRNA gene sequences for the studies of the strains were (10 mg/mL) (Promega, Madison, WI, USA), and 6 𝜇Lsterile obtained by using Eztaxon [32]. Multiple alignments of data distilled water in a final volume of 25 𝜇L. PCR was performed were performed by CLUSTAL W [34]. Evolutionary distances with Mastercycler pro (Eppendorf). Agarose gels (1% w/v) were calculated by using maximum composite likelihood were photographed after staining with ethidium bromide at −1 method and are in the units of the number of base sub- 0.5 𝜇gmL with a minivisionary imaging system. Sizes of the stitutions per site [35]. Phylogenetic tree was reconstructed fragments were estimated using the Fermentas 1 kb Plus DNA with the neighbour-joining algorithm [36]. Topology of the ladder (Fermentas, UK). resultant tree was evaluated by bootstrap analyses of the neighbour-joining dataset, based on 1000 resamplings [37]. 2.4.2. Antimicrobial Activities Test. Antimicrobial activity The sequences reported in this study have been submitted was determined by the agar cylinder diffusion method. to NCBI GenBank and the accession numbers are listed in A 6 mm diameter cylinder was taken from solid cultures appendices. and put on preseeded nutrient agar plate of the targeted microorganisms mentioned below. Up to five cylinders of different bacteria per plate were tested. Inhibition zones 2.4. Screening were expressed as diameter and measured after incubation ∘ ∘ at 37 Cfor24hforbacteriaandat28Cfor48–72hforthe 2.4.1. Primers and Molecular Screening. From the thirteen filamentous fungus and yeasts [38]. selected strains, six were subjected to molecular screening for Referencestrainsusedinthisstudywereasfollows. genes coding for putative antitumor compounds using three Sa: Staphylococcus aureus ATCC 25923, Ml: Micrococcus primer sets (Table 1). These strains were chosen on the basis luteus ATCC 9341, Ec: Escherichia coli ATCC 25922, Pa: of the presence of nonribosomal peptide synthetases/ polyke- Pseudomonas aeruginosa ATCC 27853, Ca: Candida albicans tide synthases (NRPS/PKS) genes within their genomes (clinical isolate, Algerian Central Hospital of Army of Alge- (data not published). The first set designed by Decker et al. ria), Foa: Fusarium oxysporum f. sp. albedinis afilamentous [29] amplified dNDP-glucose dehydratase genes. The second phytopathogenic fungi for date palm (Algerian National set was that of Chang and Brady [30]usedtoscreenfor Institute for Plant Protection), and Sc: Saccharomyces cere- biosynthesis of the antitumor substance BE-54017. The final visiae. set was used from the study of Ouyang et al. [31]targeting the jadomycin cyclase gene which intervenes in angucycline 2.4.3. Enzymatic Screening. Enzymaticactivities“amylolytic, production. proteolytic (caseinase), and lipolytic” were screened using 4 BioMed Research International zone clearance assays. The enzymatic substrate was incorpo- 3.4. Screening for Biotechnological Potential ratedtothemedia,andthestrainswererestreakedbyspots [39]. The tests were conducted with respect to physiological 3.4.1. Screening for Genes Coding for Putative Antitumor growth parameters of each strain. Compounds. Glu1/Glu2 primer set had 4/6 positives. High intensity band was registered for the strain Ig6. The primers targeted two different regions for the strain Bisk2. Multiple 3. Results bands were recovered from the strain GB1 while no one range 500–700 pb. PCR using this primer was negative for 3.1. Strains Isolation and Selection. Isolation plates developed thestrainA60(Figure 4(a)). The StaDVF/StaDVR primer various types of colonies. Sixty to hundred colonies were set was positive in one strain (Figure 4(b)). The PCR with found per plate in the first dilution for almost all soils, two to AuF3/AuF4 primer set was negative for all tested strains ten colonies were observed in the third dilution, and almost (Figure 4(c)). nothing in the fourth dilution plates. We have also seen that for the same dilution the number of colonies decreases when the concentration of NaCl increases. One to five colonies 3.4.2. Antimicrobial Activity. The antimicrobial activity of the which looked less represented were selected from each plate thirteen selected strains differed between strainsTable ( 2; with respect to the haloalkaliphilic character. A total of thirty- Figure 5). Among these, eight showed at least an antimi- nine isolates were distinguished. Amongst these thirty-nine crobial activity against one of the targeted microorganisms. isolates (17 were filamentous, 17 bacilli form, and 5 were cocci A highly broad spectrum antimicrobial activity inhibition form), thirteen strains—eleven with particular morphology was seen by the strain Streptomyces sp. (GB3). The strain (filamentous, which may indicate Actinobacteria that are best Bacillus sp. (Bisk4) had gram positive antibacterial activity known for the production of active biomolecules), one bacilli and antifungal activity against the filamentous fungi. The form, and one cocci form—were the subject of our study. strain Actinopolyspora sp. (M5A) inhibited the growth of The macroscopic and microscopic aspects of three of the Micrococcus luteus.TheisolateNocardia sp. (Bisk2) showed a thirteen strains are represented in Figure 2.Themolecular unique and selective activity against the yeast Saccharomyces identification by EzTaxon-e, physiological growth param- cerevisiae (Figure 5(c)). However, none of the thirteen strains eters, and enzymatic screening are described in Table 2. demonstrate specific and unique activity against the gram The alphabetical strains code used in our study refers to the negative bacteria. geographical area origin of isolation; the numerical strains code part is a simple sequential order to differentiate strains. 3.4.3. Enzymatic Activity. Strains from Bacillus and Strepto- myces were more enzymatically active and possess at least two 3.2. Physiological Growth Study. Allstrainscouldtolerateup ofthescreenedenzymes.ThestrainThermoactinomyces sp. to5%NaCl.StrainsReg1,Ker5,andHHS1wereabletotolerate (A60) was able to degrade casein and lipids. Strains Bisk2, up to 10%, whereas Bisk4 could tolerate up to 15%. Tag5 TAG5, and HHS1 seemed to have none of these screened growth started at 1% and M5A started growing at 10%; these enzymes (Figure 6). twostrainscouldgrowupto20%NaCl.Reg1,Ker5,andHHS1 are considered as halotolerant. M5A and Tag5 are considered 4. Discussion to be halophilic [40]. All strains except A60 had a versatile range of growth In this study we looked at extreme environment of the pH (5–12) indicating alkaliphilic growth; A60 (5–9) was only Algerian Sahara Desert as a source for novel strains pos- alkalitolerant. sessing interesting bioactive properties. In total, we isolated Beside the alkalitolerant character of strain A60, it pre- ∘ a collection of thirty-nine haloalkalitolerant and haloal- sented a thermophilic profile (45–60 C). With the exception kaliphilic isolates, thirteen of which were selected and of strain Bisk4, which may be considered as thermotolerant ∘ screened for genes coding for putative antitumor com- bacteria since it grows up to 55 C, the other selected bacteria pounds, as well as screening for antimicrobial and enzy- are considered to be mesophiles. matic activities. All strains were identified using 16S rRNA gene sequencing. This study represents novelty in looking 3.3. Identification. Most isolated strains belonged to the at the relatively understudied areas of Sabkha and Chott genus Streptomyces (AT1, ASB, GB1, Ig6, and GB3). The five and has yielded at least thirteen strains which poten- Actinobacteria, other than Streptomyces, were identified as tially have antitumorgenic, antimicrobial, and enzymatic follows: Reg1 and Ker5 as two different Nocardiopsis sp., HHS1 properties. as Pseudonocardia sp., M5A as Actinopolyspora sp., and Bisk2 Although often extreme and hostile ecosystems diversity as Nocardia sp.Bisk2lookslikeanewmemberasitbranches and abundance of bacteria can be low ranging from 10 to 4 out100%ofthetimefromitsnearestrelativeNocardia 10 UFC/g of soil where the physicochemical parameters are jejuensis determined by EzTaxon-e with 95% similarity for controlling factors [19], the strains retrieved and identified the 750 recovered bases. One filamentous strain A60 was in our study, in particular, of Actinobacteria strains, which identified as Thermoactinomyces sp. The bacilli Bisk4 is part of belong to various taxa, indicate a great diversity. Diversity in the Bacillus mojavensis complex and the cocci Tag5 belonged environments such as the one in this study has previously to the genus Marinococcus (Table 2; Figure 3). been investigated such as in Tunisia [9], China [41], and BioMed Research International 5 . subsp subsp. Most related species Bacillus tequilensis 10b(T) (Bacillus mojavensis group) Nocardia jejuensis N3-2(T) Streptomyces pilosus NBRC 12807(T) (Streptomyces pilosus group) Streptomyces sparsus YIM 90018(T) Nocardiopsis dassonvillei albirubida DSM 40465(T) Marinococcus halophilus DSM 20408(T) Nocardiopsis dassonvillei albirubida DSM 40465(T) Streptomyces mutabilis NBRC 12800(T) Thermoactinomyces vulgaris KCTC 9076(T) Actinopolyspora dayingensis TRM 4064(T) Pseudonocardia ammonioxydans H9(T) Streptomyces celluloflavus NBRC 13780(T) Streptomyces cyaneofuscatus JCM 4364(T) elected strains of this study. + ++ −− + +++ −−− −− −− − −− −− − −− − −− −− + + −−−− − −− − − + −−−−−−−− ++++++ −−−−−−−−− − +++++ +++++ + +++ +++ − −−−−−−−−−− −−−−−−−−−− − −− − − −−−−−−− −− −− + + ++ + −−− −−− NNN + + + ++ NNN + + NNN + NNN NNN NNN NNN + + + +++++++ Glu StaDV AuF Proteolytic Amylolytic Lipolytic Sa Ml Pa Ec Foa Ca Sc C) ∘ interval ( Temperature pH interval Growth parameters Antitumor genes Enzymatic activity Antimicrobial activity :negativeactivity,andN:nottested. − 0–5 5–12 4–42 0–5 5–12 20–42 0–5 5–12 20–40 0–5 5–9 45–60 0–7 5–12 20–37 0–7 5–12 20–37 1–20 5–12 10–42 0–15 5–12 20–55 0–12 5–12 25–30 0–10 5–12 20–42 0–10 5–12 25–42 0–10 5–12 20–42 10–20 5–12 30–40 (% g/L) Salinity interval Table 2: Physiologic characterization, antitumoral genes, enzymatic activity, antimicrobial activity, and most related species of the thirteen s Strains Bisk4 Bisk2 M5A GB1 ASB IG6 Ker5 Tag5 Reg1 AT1 HHS1 A60 GB3 +: positive activity, 6 BioMed Research International

Gx400

Gx200

(a)

Gx300 Gx200

(b)

Gx200 Gx400

(c)

Figure 2: Macroscopic morphology (left) on ISP2 and microscopic filamentous morphology (right) of three strains of this study. (a) Strain IG6: spiral chain of spores on aerial mycelium. (b) Strain Bisk2: nocardioform mycelium. (c) Strain M5A: long straight chains of spores on aerial mycelium.

previously in the Algerian Sahara soils [19, 42], which Genome sequencing followed by bioinformatics analysis has revealed that members of these extreme ecosystems forsomeofthealreadysequencedmicroorganismssuch are mainly halotolerant or halophilic organisms. Many of as Actinobacteria and Bacillus has revealed the presence of the isolated taxa in this study have previously been found several gene clusters per genome that can produce different in this environment, particularly of the Actinopolyspora, molecules [44]. Among the validly described halotolerant Nocardiopsis,andMarinococcus [9, 41–43]. Despite this and halophilic bacteria, particularly Actinobacteria,onlyfew their community structure differs both quantitatively and numbers have been subjected to analysis of their bioactive qualitatively for each different ecosystem. This would be compounds [45]. In addition, many compounds are usually due not only to the adaptation to environmental obstacles produced in very low amounts (or not at all) under typical butalsotothegeolocalisation[43], the difference of the laboratory conditions [46]. PCR based methods for specific study protocol (method, media) [41], and the sampling sites enzymes activating specific molecules are excellent screening [42]. toolsforthesestrains;theywouldnotonlyindicatethe BioMed Research International 7

68 Strain GB3 (JQ690542) 72 Streptomyces cyaneofuscatus; JCM 4364 (AY999770) 54 Strain GB1 (JQ690543) 41 Streptomyces cellulofavus; NBRC 13780 (AB184476) 70 Strain Ig6 (JQ690545) 98 Streptomyces sparsus; YIM 90018 (AJ849545) Strain ASB (JQ690544) Streptomyces mutabilis; NBRC 12800 (AB184156) 95 Strain AT1 (JQ690546) 71 100 Streptomyces pilosus; NBRC 12807 (AB184161) Strain Ker5 (JQ690548) 100 Strain Reg1 (JQ690549) Nocardiopsis dassonvillei subsp. albirubida; DSM 40465 (X97882) 52 100 Strain M5A (KJ409655) Actinopolyspora dayingensis; TRM 4064 (KC461229) Strain HHS1 (JQ690547) 78 100 Pseudonocardia ammonioxydans; H9 (AY500143) 57 Strain Bisk2 (JQ690551) 98 Nocardia jejuensis; N3-2 (AY964666) 100 Strain Bisk4 (JQ690553) Bacillus tequilensis; 10b (HQ223107) Strain A60 (JQ690550) 99 99 Termoactinomyces vulgaris; KCTC 9076 (AF138739) 57 Strain Tag5 (JQ690552) 100 Marinococcus halophilus; DSM 20408 (X90835) Escherichia coli strain KCTC 2441 (EU014689)

0.02

Figure 3: Molecular phylogeny of thirteen selected bacteria and the most related type strains species using partial 16S rRNA sequences. The evolutionary distances were computed using the maximum composite likelihood method and are in the units of the number ofbase substitutions per site. Tree topology was constructed using MEGA 4.0. Bootstrap values (𝑛 = 1000 replicates) were indicated at the nodes. Escherichia coli KCTC2441 sequence was added as an out group for this tree. M Bisk2 GB1 W Ig6 M5A Bisk4 A60 GB1 Ig6 M Bisk2 Bisk4 A60 W M5A W GB1 Bisk4 A60 Ig6 M5A M Bisk2

700 700 700 500 500 500

(a) (b) (c)

Figure 4: Agarose gel electrophoresis of PCR products from genomic DNA of six strains of the present study with selective fragments amplification range 500–700 bp using primers: (a) Glu1/Glu2, (b) StaDVF/StaDVR, and (c) AuF3/AuF4. M: 1 kb Plus DNA ladder; W:water control. 8 BioMed Research International

ASB Bisk4 GB3

2 Bisk4 Bisk GB3 Foa

Bisk2

(a) (b) (c)

Figure 5: Antimicrobial activity of some strains among the selected strains: (a) antibacterial activity against Staphylococcus aureus,(b) antifungal activity against Fusarium oxysporum f. sp. albedinis, and (c) antifungal activity of the strain Nocardia sp. (Bisk2) against the yeast Saccharomyces cerevisiae.

ASB

AT1 60 A Ig6 ASB

Ker5 GB3 Reg1

(a) (b) (c)

Figure 6: Enzymatic activities of some strains among the selected strains. (a) Proteases (caseinase), (b) lipases, and (c) amylases.

presence of probable genes clusters but also help in bio- The primer set has also been reported in other screening chemical characterisation of the molecules. These methods studiesfortalosinsAandBcluster,anantifungal[54], for would help in reducing the number of strains that need to caprazamycin biosynthesis, an antimycobacterial [55], and be screened by cultural methods. The PCR based methods more recently we have used this set to screen for amicetin not only are limited to genomic DNA but also can be applied biosynthesis gene cluster, an antibacterial and antiviral agent for the screening of eDNA that lead to the discovery of new [56]. The second primer set was designed by Chang and active biomolecules [30]. Screening for potential production Brady [30] who screened a previously archived soil eDNA of a particular type of biomolecules such as antibiotics and cosmid library by PCR using degenerate primers designed antitumorales, without going through the tedious biochem- to recognize conserved regions in known oxytryptophan istry process, is more efficient when the typing protocol dimerization genes (StaD/RebD/VioB etc). The oxytrypto- is targeting the biosynthesis gene cluster rather than the phan dimerization enzymes were chosen as probes because taxonomic marker genes (e.g., 16S rRNA gene) which often this enzyme family is used in the biosynthesis of structurally give misleading results [47, 48]. diverse tryptophan dimmers, which have shown an antitu- In our study, we have been interested in molecular moral activity. Both indolocarbazole biosynthetic gene clus- screening of bioactive genes coding for putative antitu- ters (e.g., staurosporine, rebeccamycin, K-252a, and AT2433) mor compounds. The degenerate primers Glu1/Glu2 for and violacein biosynthetic gene clusters contain homologous the conserved N-terminal sequence of dNDP-glucose 4,6- enzymesthatcarryouttheoxidation(StaO/RebO/VioA)and dehydratase genes have been extensively used to screen subsequent dimerization (StaD/RebD/VioB)oftryptophan. out for clusters of active biomolecules with antitumoral One among the six screened strains was positive for the set activity such as novobiocin [49], enediyne [50], elloramycin oftheprimers,strainM5A.Thiswouldsignifythatthestrain [51], sibiromycin [52], ravidomycin, and chrysomycin [53]. M5A could produce tryptophan dimmers compound(s). BioMed Research International 9

The sequencing result followed by blast for the PCR prod- Appendix ucts of M5A using StaDVF/StaDVR primers set (GenBank: KJ560370) has shown 76% homology to the uncultured bac- GenBank accession numbers for 16S RNA gene sequences of terium clone AR1455 rebeccamycin-like tryptophan dimer 13 strains of this study are GB3 (JQ690542), GB1 (JQ690543), gene cluster (GenBank: KF551872) that was studied by Chang ASB (JQ690544), Ig6 (JQ690545), AT1 (JQ690546), HHS1 and Brady [30], while, for the strains Streptomyces sp.Ig6,it (JQ690547), Ker5 (JQ690548), Reg1 (JQ690549), A60 has shown a mixed PCR product; we think this is probably (JQ690550), Bisk2 (JQ690551), Tag5 (JQ690552), Bisk4 (JQ690553), and M5A (KJ409655). due to the presence of multiple variable copies of this gene in this strain. The different patterns of activity against the targeted Conflict of Interests microorganisms observed in this study may indicate a vari- The authors declare that there is no conflict of interests ety of the produced active biomolecules. The antimicrobial regarding the publication of this paper. activity of Bisk2, most closely related to Nocardia jejuensis [57], has never been reported to our knowledge. This result encourages us to consider Bisk2 as probably a new member or Acknowledgments at least a new strain of Nocardia.Genomesequencing,DNA- The authors acknowledge Warwick University staff, in par- DNA hybridising, and molecular chemotaxonomy would ticular Dr. Calvo-Bado A. L., Dr. Khalifa A., and Dr. Wit- give more knowledge about its taxonomic position among the comb L. The authors also acknowledge Professor Naim M. Nocardia species. from HCA, Dr. Antri K. USTHB for providing the targeted The Sahara Desert is subject to large fluctuations in microorganisms, and Mr. Bouhzila F. from environmental parameters such as temperature, pH, or salinity. It is popu- Biotechnology, Polytechnical School, Algiers, for physical- lated by communities of organisms with intrinsic genomic chemical soils parameters determination. The authors give heterogeneity for adaptation. The mechanisms of cell adapta- special thanks to Mr. Mohammed A., Mr. Slama G., and Mr. tion engage several enzymatic processes that may be a source Nateche` M. for the help. The authors would also like to thank of enzymes that show a higher level of stability and activity the anonymous reviewers for the analysis and the enrichment over a wider range of conditions. The screened enzymes of this paper. In the end, the authors would like to thank found in this study (proteases, amylases, and lipases) would the Algerian Ministry of Higher Education and Scientific be economically valuable since they were screened from such Research and the University of Warwick for supporting this environments and are likely to exhibit rare properties; these work. CB has received funding from the European Union’s extremozymes are of great value to biotechnology industries Seventh Framework Programme for research, technological [7, 58, 59]. development, and demonstration under Grant no. 289285.

References 5. Conclusion [1] M. Podar and A. L. Reysenbach, “New opportunities revealed Exploration of biodiversity and biotechnological potential by biotechnological explorations of extremophiles,” Current of desert microorganisms has gone several steps forward Opinion in Biotechnology,vol.17,no.3,pp.250–255,2006. in recent years. The Sahara Desert is one of the biggest [2] S. Subramaniam, “Productivity and attrition: key challenges for worldwide. It spreads upon several countries of Africa. biotech and pharma,” Drug Discovery Today,vol.8,no.12,pp. These countries are among the countries worldwide to have 513–515, 2003. the smallest registration rates of biodiversity in biological [3]K.S.Lam,“Newaspectsofnaturalproductsindrugdiscovery,” databases [60]. Trends in Microbiology,vol.15,no.6,pp.279–289,2007. In addition to the insights on the biodiversity of Algerian [4] N. Peric-Concha´ and P. F. Long, “Mining the microbial Sahara Desert, to our knowledge, this is the first time to use metabolome: a new frontier for natural product lead discovery,” the molecular screening of these genes coding for putative Drug Discovery Today,vol.8,no.23,pp.1078–1084,2003. antitumor compounds to analyse Algerian strains. In this [5] J. Sp´ızek,ˇ J. Novotna,´ T. Rezanka,ˇ and A. L. Demain, “Do we study, we have highlighted the interesting presence of diverse need new antibiotics? The search for new targets and new compounds,” Journal of Industrial Microbiology and Biotechnology, haloalkalitolerant and haloalkaliphilic strains with potential vol. 37, no. 12, pp. 1241–1248, 2010. antitumorigenic, bioactive, and other interesting enzymes. [6] F. A. Rainey and A. Oren, “Extremophile microorganisms and Future work will concentrate on more cloning and sequenc- the methods to handle them,” in Extremophiles Methods in ing for whole clusters, chemical characteristics, identification Microbiology,F.A.RaineyandA.Oren,Eds.,vol.35,pp.1–25, by application of mass spectrum, and other enzymatic and Academic Press, London, UK, 2006. biochemical techniques that would be more suitable for better [7]Y.Ma,E.A.Galinski,W.D.Grant,A.Oren,andA.Ventosa, determination of the nature of the elaborated compounds “Halophiles 2010: life in saline environments,” Applied and produced by the strains identified in this study particu- Environmental Microbiology, vol. 76, no. 21, pp. 6971–6981, 2010. larly of Nocardia sp. Bisk2, Actinopolyspora sp. M5A, and [8] L. J. Rothschild and R. L. Mancinelli, “Life in extreme environ- Streptomyces sp. Ig6. ments,” Nature,vol.409,no.6823,pp.1092–1101,2001. 10 BioMed Research International

[9] D. El Hidri, A. Guesmi, A. Najjari et al., “Cultivation-dependant [26] S. Boutaiba, T. Bhatnagar, H. Hacene, D. A. Mitchell, and J. C. assessment, diversity, and ecology of haloalkaliphilic bacteria Baratti, “Preliminary characterisation of a lipolytic activity from in arid saline systems of southern Tunisia,” BioMed Research an extremely halophilic archaeon, Natronococcus sp.,” Journal International, vol. 2013, Article ID 648141, 15 pages, 2013. of Molecular Catalysis B: Enzymatic,vol.41,no.1-2,pp.21–26, [10] G. S. H. Soliman and H. G. Trueper, “Halobacterium pharaonis 2006. sp.nov., a new, extremely haloalkaliphilic archaebacterium with [27] E. B. Shirling and D. Gottlieb, “Methods for characterization low magnesium requirement,” Zentralblatt fur¨ Bakteriologie of Streptomyces species,” International Journal of Systematic Mikrobiologie und Hygiene: I. Abt. Originale C,vol.3,no.2,pp. Bacteriology,vol.16,pp.313–340,1966. 318–329, 1982. [28]U.Edwards,T.Rogall,H.Blocker,M.Emde,andE.C.Bottger, [11] K. Horikoshi, Alkaliphiles,WileyOnlineLibrary,2008. “Isolation and direct complete nucleotide determination of [12] K. Horikoshi, “Alkaliphiles: some applications of their prod- entire genes. Characterization of a gene coding for 16S riboso- ucts for biotechnology,” Microbiology and Molecular Biology mal RNA,” Nucleic Acids Research,vol.17,no.19,pp.7843–7853, Reviews,vol.63,no.4,pp.735–750,1999. 1989. [13] H. Takami, Y. Takaki, and I. Uchiyama, “Genome sequence [29] H. Decker, S. Gaisser, S. Pelzer et al., “A general approach for of Oceanobacillus iheyensis isolated from the Iheya Ridge and cloning and characterizing dNDP-glucose dehydratase genes its unexpected adaptive capabilities to extreme environments,” from actinomycetes,” FEMS Microbiology Letters,vol.141,pp. Nucleic Acids Research, vol. 30, no. 18, pp. 3927–3935, 2002. 195–201, 1996. [14] S. P. Singh, “Extreme environments and extremophiles,” in E- [30] F. Y. Chang and S. F. Brady, “Cloning and characterization of Book: Environmental Microbiology, pp. 1–35, National Science an environmental DNA-derived gene cluster that encodes the Digital Library (CSIR), New Delhi, India, 2006. biosynthesis of the antitumor substance BE-54017,” Journal of [15] A.A.Joshi,P.P.Kanekar,A.S.Kelkaretal.,“Cultivablebacterial the American Chemical Society, vol. 133, no. 26, pp. 9996–9999, diversity of alkaline Lonar lake, India,” Microbial Ecology,vol. 2011. 55,no.2,pp.163–172,2008. [31]Y.Ouyang,H.Wu,L.Xieetal.,“Amethodtotypethepotential [16] H. Hacene,N.Sabaou,N.Bounaga,andG.Lefebvre,“Screening` angucycline producers in actinomycetes isolated from marine for non-polyenic antifungal antibiotics produced by rare Acti- sponges,” International Journal of Sciences: Basic and Applied nomycetales,” Microbios,vol.79,no.319,pp.81–85,1994. Research,vol.99,no.4,pp.807–815,2011. [17] H. Hacene, K. Kebir, D. S. Othmane, and G. Lefebvre, “HM17, a [32] O. S. Kim, Y. J. Cho, K. Lee et al., “Introducing EzTaxon-e: a new polyene antifungal antibiotic produced by a new strain of prokaryotic 16S rRNA Gene sequence database with phylotypes Spirillospora,” Journal of Applied Bacteriology,vol.77,no.5,pp. that represent uncultured species,” International Journal of 484–489, 1994. Systematic and Evolutionary Microbiology, vol. 62, part 3, pp. [18] H. Hacene,` F. Daoudi-Hamdad, T. Bhatnagar, J. C. Baratti, and 716–721, 2012. G. Lefebvre, “H107, a new aminoglycoside anti-Pseudomonas [33]K.Tamura,J.Dudley,M.Nei,andS.Kumar,“MEGA4:Molec- antibiotic produced by a new strain of Spirillospora,” Microbios, ular Evolutionary Genetics Analysis (MEGA) software version vol.102,no.402,pp.69–77,2000. 4.0,” Molecular Biology and Evolution,vol.24,no.8,pp.1596– [19] H. Hacene,ˇ F. Rafa, N. Chebhouni et al., “Biodiversity of 1599, 2007. prokaryotic microflora in El Golea Salt lake, Algerian Sahara,” [34] J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTAL Journal of Arid Environments,vol.58,no.3,pp.273–284,2004. W: improving the sensitivity of progressive multiple sequence [20] S. Boutaiba, H. Hacene, K. A. Bidle, and J. A. Maupin-Furlow, alignment through sequence weighting, position-specific gap “Microbial diversity of the hypersaline Sidi Ameur and Himalatt penalties and weight matrix choice,” Nucleic Acids Research,vol. Salt Lakes of the Algerian Sahara,” Journal of Arid Environments, 22, no. 22, pp. 4673–4680, 1994. vol.75,no.10,pp.909–916,2011. [35]K.Tamura,M.Nei,andS.Kumar,“Prospectsforinferring [21] A. Bouanane-Darenfed, M. L. Fardeau, P. Gregoire´ et al., very large phylogenies by using the neighbor-joining method,” “Caldicoprobacter algeriensis sp. nov. a new thermophilic anaer- Proceedings of the National Academy of Sciences of the United obic, xylanolytic bacterium isolated from an Algerian hot States of America,vol.101,no.30,pp.11030–11035,2004. spring,” Current Microbiology,vol.62,no.3,pp.826–832,2011. [36] N. Saitou and M. Nei, “The neighbor-joining method: a new [22] A. N. Addou, P. Schumann, C. Sproer,¨ H. Hacene, J. L. Cayol, method for reconstructing phylogenetic trees,” Molecular Biol- andM.L.Fardeau,“Melghirimyces algeriensis gen. nov., sp. nov., ogy and Evolution,vol.4,no.4,pp.406–425,1987. amemberofthefamilyThermoactinomycetaceae,isolatedfrom [37] J. Felsenstein, “Confidence limits on phylogenies: an approach asaltlake,”International Journal of Systematic and Evolutionary using the bootstrap,” Evolution,vol.39,pp.783–791,1985. Microbiology,vol.62,part7,pp.1491–1498,2012. [38] R. Forar-Laidi, A. Abderrahmane, and A. A. Hocine-Norya, [23] A. N. Addou, P. Schumann, C. Sproer¨ et al., “Melghirimyces “Identification and antibiosis of a novel actinomycete strain thermohalophilus sp. nov., a thermoactinomycete isolated from RAF-11 isolated from Iraqi soil,” International Journal of Sci- an Algerian salt lake,” International Journal of Systematic and ences: Basic and Applied Research,vol.12,no.1,pp.141–159,2013. Evolutionary Microbiology,vol.63,no.part5,pp.1717–1722, [39] E. Phillips and P. Nash, “Culture media,” in Manual of Clinical 2013. Microbiology,E.H.Lennette,A.Balows,W.J.HauslerJr., [24] M. Amziane, F. Metiaz, A. Darenfed-Bouanane et al., “Vir- andH.J.Shadomy,Eds.,pp.1064–1065,AmericanSocietyfor gibacillus natechei sp. nov., a moderately halophilic bacterium Microbiology, Washington, DC, USA, 4th edition, 1985. isolatedfromsedimentofasalinelakeinsouthwestofAlgeria,” [40] A. Ventosa, E. Mellado, C. Sanchez-Porro, and M. C. Marquez, Current Microbiology,vol.66,no.5,pp.462–466,2013. “Halophilic and halotolerant micro-organisms from soils,” in [25] T. Bhatnagar, S. Boutaiba, H. Hacene et al., “Lipolytic activity Microbiology of Extreme Soils, Soil Biology,P.DionandC.S. from Halobacteria: screening and hydrolase production,” FEMS Nautiyal, Eds., vol. 13, pp. 87–115, Springer, Berlin, Germany, Microbiology Letters,vol.248,no.2,pp.133–140,2005. 2008. BioMed Research International 11

[41]J.Wu,T.Guan,H.Jiangetal.,“Diversityofactinobacterial [56] G. Zhang, H. Zhang, S. Li et al., “Characterization of the community in saline sediments from Yunnan and Xinjiang, amicetin biosynthesis gene cluster from Streptomyces vinaceus- China,” Extremophiles, vol. 13, no. 4, pp. 623–632, 2009. drappus NRRL 2363 implicates two alternative strategies for [42] A. Meklat, N. Sabaou, A. Zitouni, F. Mathieu, and A. Lebrihi, amide bond formation,” Applied and Environmental Microbiol- “Isolation, taxonomy, and antagonistic properties of halophilic ogy,vol.78,no.7,pp.2393–2401,2012. actinomycetes in saharan soils of algeria,” Applied and Environ- [57] S. D. Lee, “Nocardia jejuensis sp. nov., a novel actinomycete mental Microbiology,vol.77,no.18,pp.6710–6714,2011. isolated from a natural cave on Jeju Island, Republic of Korea,” [43] E. Pagaling, H. Wang, M. Venables et al., “Microbial biogeog- International Journal of Systematic and Evolutionary Microbiol- raphy of six salt lakes in Inner Mongolia, China, and a salt lake ogy,vol.56,no.3,pp.559–562,2006. in Argentina,” AppliedandEnvironmentalMicrobiology,vol.75, [58] R. Margesin and F. Schinner, “Potential of halotolerant and no. 18, pp. 5750–5760, 2009. halophilic microorganisms for biotechnology,” Extremophiles, [44] “Genomes OnLine Database,” http://genomesonline.org/cgi- vol. 5, no. 2, pp. 73–83, 2001. bin/GOLD/index.cgi. [59] A. Oren, “Industrial and environmental applications of [45] J. Hamedi, F. Mohammadipanah, and A. Ventosa, “Systematic halophilic microorganisms,” Environmental Technology,vol.31, and biotechnological aspects of halophilic and halotolerant no. 8-9, pp. 825–834, 2010. actinomycetes,” Extremophiles,vol.17,pp.1–13,2013. [60]O.Selama,P.James,F.Nateche,E.M.H.Wellington,andH. [46] A. K. Chaudhary, D. Dhakal, and J. K. Sohng, “An insight into Hacene,` “The world bacterial biogeography and biodiversity the “-omics” based engineering of streptomycetes for secondary through databases: a case study of NCBI Nucleotide Database metabolite overproduction,” BioMed Research International, and GBIF Database,” BioMed Research International,vol.2013, vol.2013,ArticleID968518,15pages,2013. Article ID 240175, 11 pages, 2013. [47] M. Metsa-Ketel¨ a,¨ L. Halo, E. Munukka, J. Hakala, P. Mants¨ ala,¨ and K. Ylihonko, “Molecular evolution of aromatic polyketides and comparative sequence analysis of polyketide ketosynthase and 16S ribosomal DNA genes from various Streptomyces species,” AppliedandEnvironmentalMicrobiology,vol.68,no. 9, pp. 4472–4479, 2002. [48] C. P. Ridley, Y. L. Ho, and C. Khosla, “Evolution of polyketide synthases in bacteria,” Proceedings of the National Academy of Sciences of the United States of America,vol.105,no.12,pp.4595– 4600, 2008. [49] M. Steffensky, A.uhlenweg,Z.X.Wang,S.M.Li,and M¨ L. Heide, “Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891,” Antimicrobial Agents and Chemotherapy,vol.44,no.5,pp.1214–1222,2000. [50] W. Liu and B. Shen, “Genes for production of the enediyne antitumor antibiotic C-1027 in Streptomyces globisporus are clustered with the cagA gene that encodes the C-1027 apopro- tein,” Antimicrobial Agents and Chemotherapy,vol.44,no.2,pp. 382–392, 2000. [51] A. Ramos, F. Lombo,´ A. F. Brana,˜ J. Rohr, C. Mendez,´ and J. A. Salas, “Biosynthesis of elloramycin in Streptomyces olivaceus requires glycosylation by enzymes encoded outside the aglycon cluster,” Microbiology,vol.154,no.3,pp.781–788,2008. [52] W. Li, A. Khullar, S. Chou, A. Sacramo, and B. Gerratana, “Biosynthesis of sibiromycin, a potent antitumor antibiotic,” AppliedandEnvironmentalMicrobiology,vol.75,no.9,pp. 2869–2878, 2009. [53] M. K. Kharel, S. E. Nybo, M. D. Shepherd, and J. Rohr, “Cloning and characterization of the ravidomycin and chrysomycin biosynthetic gene clusters,” ChemBioChem, vol. 11, no. 4, pp. 523–532, 2010. [54] T. M. Yoon, J. W. Kim, J. G. Kim, W. G. Kim, and J. W. Suh, “Talosins A and B: new isoflavonol glycosides with potent antifungal activity from Kitasatospora kifunensis MJM341 I. Taxonomy, fermentation, isolation, and biological activities,” Journal of Antibiotics,vol.59,no.10,pp.633–639,2006. [55] L. Kaysser, E. Wemakor, S. Siebenberg et al., “Formation and attachment of the deoxysugar moiety and assembly of the gene cluster for caprazamycin biosynthesis,” Applied and Environmental Microbiology,vol.76,no.12,pp.4008–4018, 2010. Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 924235, 9 pages http://dx.doi.org/10.1155/2014/924235

Research Article Absence of Cospeciation between the Uncultured Frankia Microsymbionts and the Disjunct Actinorhizal Coriaria Species

Imen Nouioui,1 Faten Ghodhbane-Gtari,1 Maria P. Fernandez,2 Abdellatif Boudabous,1 Philippe Normand,2 and Maher Gtari1,3

1 Laboratoire Microorganismes et Biomolecules´ Actives, UniversitedeTunisElManar(FST)etUniversit´ e´ Carthage (INSAT), 2092 Tunis, Tunisia 2 Ecologie Microbienne, Centre National de la Recherche Scientifique UMR 5557, Universite´ Lyon I, 69622 Villeurbanne Cedex, France 3 Laboratoire Microorganismes et Biomolecules´ Actives, Faculte´ des Sciences de Tunis, Campus Universitaire, 2092 Tunis, Tunisia

Correspondence should be addressed to Maher Gtari; [email protected]

Received 4 March 2014; Revised 25 March 2014; Accepted 27 March 2014; Published 22 April 2014

Academic Editor: Ameur Cherif

Copyright © 2014 Imen Nouioui et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Coriaria is an actinorhizal plant that forms root nodules in symbiosis with nitrogen-fixing actinobacteria of the genus Frankia.This symbiotic association has drawn interest because of the disjunct geographical distribution of Coriaria in four separate areas of the world and in the context of evolutionary relationships between host plants and their uncultured microsymbionts. The evolution of Frankia-Coriaria symbioses was examined from a phylogenetic viewpoint using multiple genetic markers in both bacteria and host-plant partners. Total DNA extracted from root nodules collected from five species: C. myrtifolia, C. arborea, C. nepalensis, C. japonica,andC. microphylla, growing in the Mediterranean area (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively, was used to amplify glnA gene (glutamine synthetase), dnaA gene (chromosome replication initiator), and the nif DK IGS (intergenic spacer between nifD and nifK genes) in Frankia and the matK gene (chloroplast-encoded maturase K) and the intergenic transcribed spacers (18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA) in Coriaria species. Phylogenetic reconstruction indicated that the radiations of Frankia strains and Coriaria species are not congruent. The lack of cospeciation between the two symbiotic partners may be explained by host shift at high taxonomic rank together with wind dispersal and/or survival in nonhost rhizosphere.

1. Introduction association with Betulaceae, Myricaceae, and Casuarinaceae. Cluster 2 contains Frankia nodulating species from the The genus Frankia comprises nitrogen-fixing actinobac- Coriariaceae, Datiscaceae, and Rosaceae families as well as teria that are able to induce perennial root nodules on Ceanothus of the Rhamnaceae. Frankia strains in cluster 3 woody dicotyledonous plants called actinorhizals [1]. The form effective root nodules on plants from members of the actinorhizal plant families belong to three dicotyledonous Myricaceae, Rhamnaceae, Elaeagnaceae, and Gymnostoma of orders: Fagales (Betulaceae,Casuarinaceae, and Myricaceae), the Casuarinaceae. Rosales (Elaeagnaceae, Rhamnaceae, and Rosaceae), and Symbiotic Frankia strains have been only isolated from Cucurbitales (Coriariaceae and Datiscaceae) [2]. Analysis of Fagales (Frankia cluster 1) and the families Elaeagnaceae the molecular phylogeny of members of Frankia genus con- and Rhamnaceae (Frankia cluster 3) of the Rosales, while sistently identifies four main clusters regardless of the typing Frankia of cluster 2 have still not yet been isolated in culture locus used [3]. Three symbiotic Frankia clusters containing despite repeated attempts [2]. The position in the Frankia strains able to establish effective nodules and fulfill Koch’s phylogenetic tree of cluster 2 relative to the other clusters has postulates and one atypical with strains unable to establish varied depending on the marker used. It was proposed at the effective nodulation on their host plants have been defined base using glnA and 16S rRNA genes [4, 5], derived with ITS among Frankia genera. Cluster 1 includes Frankia strains in 16S–23S rRNA genes [6] and concatenated gyrB, nif Hand 2 BioMed Research International glnII genes [7] and should be clarified by the upcoming whole Benbrahim (University of Fes, Fes, Morocco), Dr. Takashi genome phylogeny. Nevertheless, a position at the base of all Yamanaka (Forest and Forestry Products Research Institute, symbiotic lineages has been retained in the latest treatment of Ibaraki, Japan), and Dr. Jean-Claude Cleyet-Marel (INRA- Bergey’s manual [8]. IRD, Montpellier, France). Individual lobes were selected, Cross-inoculation studies using crushed nodules suggest surface-sterilized in 30% (vol/vol) H2O2, and rinsed several that cluster 2 strains form a separate and unique host times with distilled sterile water. The DNA extraction from specificity group9 [ –11], even though provenances from the single nodule lobes was performed as previously described full geographical range have not yet been tested. Despite byRouvieretal.[26]. Nodule lobes were crushed with sterile the high taxonomic diversity of host plants belonging to plastic mortars and pestles in 300 𝜇L of extraction buffer the cross-inoculation group of cluster 2 and its disjunct (100 mM Tris (pH 8), 20 mM EDTA, 1.4 M NaCl, 2% (wt/vol) range, uncultured Frankia in root nodules of several host CTAB (cetyltrimethyl ammonium bromide), and 1% (wt/vol) plants have so far shown a low level of diversity regardless PVPP (polyvinyl polypyrrolidone)). The homogenates were ∘ of the typing locus used [6, 7, 11–16], suggesting a recent incubated at 65 C for 60min, extracted with chloroform- emergence, a strong and recent evolutionary bottleneck, or isoamyl alcohol (24 : 1, vol/vol) and the resulting DNA was a nonrepresentative sampling. The time of emergence of all ethanol-precipitated and resolubilized. The extracted DNA Frankia lineages is poorly documented as no convincing was used for PCR amplification of both bacterial and plant fossil remains. An equivalence between 16S rRNA sequences DNA regions using the primers listed in Table 2. The ampli- distance and time of emergence has been proposed by cons were then cycle-sequenced in both directions using Ochman and Wilson [17] where 1% is equivalent to 50 million an ABI cycle sequencing kit (Applied Biosystem 3130). The years, and since 4% divergence exists between Frankia cluster nucleotide sequences obtained in this study were deposited in 2 and the other clusters, one would conclude that Frankia theNCBInucleotidesequencedatabaseundertheaccession emerged 200 million years ago [5], which would mean that numbers given in Table 1. there is missing diversity either due to a recent evolutionary bottleneck or due to a lack of sampling [16]. A possibility thus 2.2. Phylogenetic Analysis. Frankia strain CcI3 and Casuarina exists that the missing variability in cluster 2 strains is due to equisetifolia were used as outgroups in this study because the fact that sampling has so far been limited essentially to they are physiologically distinct from the group studied yet North American and Mediterranean areas. phylogenetically close. The data sets were completed with Evidence for cospeciation has been found so far only in homologous sequences present in the databases (Table 1). the case of Casuarina species growing in Australia and their Alignments of Frankia glnA, dnaA, and IGS nif D-K and Cori- Frankia [18]thatareintheirimmensemajorityresistantto aria matK and 18S rRNA-ITS1-5.8S rRNA-ITS2-28S rRNA growth in pure culture. Among actinorhizal plants of the were generated with ClustalW [27], manually edited with Cucurbitales subclade, the family Coriariaceae, with only one MEGA 5.0 [28]. Bacterial and plant sequences were separately genus, Coriaria, contains about 17 species [19]thatoccurin concatenated and then used to examine maximum-likelihood four disjunct areas of the world: the Mediterranean, Southeast cladogram evolutionary relationships of each symbiotic part- Asia, Central and South America, and the Pacific islands of ner using 1000 bootstraps by following the GTR + G base New Zealand and Papua New Guinea [20–24]. Yokoyama et substitution model. The distance between the sequences was al. [19]consideredthattheEurasianspeciesarebasalandhave calculated using Kimura’s two-parameter model [29]. Phy- emerged some 60 million years ago. This date is in agreement logenetic trees were constructed using the Neighbor-Joining with the 65 million years proposed by Bell et al. [25]basedon method [30] with 1000 bootstraps [31]asimplementedin multiple genes (rbcL, 18S rDNA, atpB) phylogeny, while the MEGA 5.0. In parallel, a Bayesian inference was realized same authors propose an emergence of the Casuarinaceae at with MrBayes [32] using the GTR + G model and 1,000,000 about 30 million years. generations. The present study was aimed at testing the hypothesis A statistical test for the presence of congruence between of cospeciation between uncultured Frankia microsymbionts Coriaria and Frankia phylogenies was evaluated through and their Coriaria host species sampled from sites covering global distance-based fitting in ParaFit program33 [ ]as the full geographical range of the genus: Coriaria myrtifolia implemented in CopyCat [34] and tests of random associa- (Morocco and France), C. nepalensis (Pakistan), C. arborea tion were performed with 9999 permutations globally across (New Zealand), C. japonica (Japan), and C. microphylla both phylogenies for each association. (Mexico). An additional statistical test for correlation between geographical distances (obtained using http://www.daftlogic 2. Materials and Methods .com/projects-google-maps-distance-calculator.htm)and phylogenetic distances was made using Pearson’s r correlation 2.1. DNA Extraction, PCR Amplification, and Sequencing. implemented in the R software [35]. Root nodules from naturally occurring Coriaria species (Table 1) were kindly provided by Dr. Mar´ıa Valdes´ (Escuela 3. Results Nacional de Ciencias Biologicas,´ Mexico,´ DF, Mexico),´ Dr. Sajjad Mirza (National Institute for Biotechnology Genetic To avoid taxonomic ambiguities, DNAs from both Coriaria Engineering, Faisalabad, Pakistan), Dr. Warwick Silvester hosts and Frankia microsymbionts were characterized on the (University of Waikato, Waikato, New Zealand), Dr. Kawther same root nodule tissues. The method of DNA isolation from BioMed Research International 3 ]) ]) 19 19 This study This study This study This study This study This study This study This study This study This study This study This study This study References D-K — nif KC796561 KC796557 KC796578 KC796558 KC796563 KC796579 KC796565 KC796556 KC796562 KC796567 KC796580 KC796566 AIGS dna KC796593 KC796583 KC796585 KC796595 KC796592 KC796588 KC796589 KC796505 KC796507 KC796594 KC796590 KC796584 KC796506 522 KC796582 KC796555 This study A gln KC796533 KC796532 KC796535 KC796523 KC796525 KC796539 KC796538 KC796528 KC796524 KC796529 KC796530 KC796534 KC796540 K mat AB016459 et (Yokoyama al., 2000 [ AB016456 et (Yokoyama al., 2000 [ AF280102 Yang et al., unpublished AF280101 Yang et al., unpublished AF280103 Yang et al., unpublished ITS1-ITS2 Plant sequence accession number Bacterial sequence accession number root nodules and sequences used in this study. CjJE CjJB KC796594 KC796537 KC796504 KC796577 This study CjJA KC796605 KC796536 KC796503 KC796576 This study CjJC CjJD CnP1 KC796597 KC796607 KC796544 KC796508 KC796584 This study CnP3 KC796546 KC796510 KC796586 This study CnP2 KC796545 KC796509 KC796585 This study CnP4 CmF1 KC796526 KC796586 KC796559 This study Coriaria CmF3 CmF5 CmF2 KC796593 KC796602 KC796527 KC796587 KC796560 This study CmF4 CmNy1 KC796598 KC796603 KC796531 KC796591 KC796564 This study CmMs1 KC796592 KC796601 KC796 CmNy3 CmNy5 CmNy2 CmM1c KC796519 KC796580 KC796552 This study CmM1a KC796590 KC796599 KC796517 KC796578 KC796550 This study CmNy4 CmMs3 CmMs2 CmM1b KC796518 KC796579 KC796551 This study CmMs4 CmM2a KC796591 KC796600 KC796520 — KC796553 This study CmM2b KC796521 KC796581 KC796554 This study Nodule labels /10 m 󸀠󸀠 N ∘ Table 1: List of E/41 m 󸀠󸀠 42, 89 󸀠 E/259 m 27 󸀠󸀠 23.97 ∘ 󸀠 E/33.9042 󸀠󸀠 52 ∘ 21.82 25 󸀠 󸀠 ,+133 󸀠󸀠 N/3 08 23 ∘ ∘ 󸀠󸀠 E/1290 m 󸀠󸀠 39.18 N/5 󸀠 N73 󸀠󸀠 51.48 092 565E/140 m 󸀠 󸀠 󸀠󸀠 45 󸀠 ∘ 15 36 58 󸀠 20 ∘ ∘ ∘ 46.50 54 󸀠 ∘ 21 ∘ E/2291.2 m ∘ 979N/04 879N/05 󸀠 󸀠 01 00 ∘ ∘ Bab Berred, Chefchaouen: 35 France Nyons, 44 Montpellier, 43 Morocco Oued El Koub, Ouezzane: 35 Japan Tosa district, +33 Pakistan Murree, +33 73.3903 Species Locality coordinates/altitude (asl) C. myrtifolia C. japonica C. nepalensis 4 BioMed Research International ]) ]) ]) ]) ]) ]) ]) ]) 19 19 19 19 19 19 51 50 unpublished References D-K nif AIGS dna A gln CP002801 CP002801 CP002801 (Persson et al., 2011 [ CP000249 CP000249 CP000249 (Normand et al., 2007 [ K mat AB16454 (Yokoyama et al., 2000 [ AB016461 et (Yokoyama al., 2000 [ AB016455 et (Yokoyama al., 2000 [ AB015462 Sogo et al., unpublished AB016458 et (Yokoyama al., 2000 [ AB016462 et (Yokoyama al., 2000 [ AF485250 Forrest and Hollingsworth AB016464 et (Yokoyama al., 2000 [ AY091813 Yang et al., unpublished AY091815 Yang et al., unpublished AY091817 Yang et al., unpublished AY091814 Yang et al., unpublished AY091816 Yang et al., unpublished EF635475 Rotherham et al., unpublished EF635457 Rotherham et al., unpublished AF280100 Yang et al., unpublished ITS1-ITS2 AF280104 Yang et al., unpublished AF277293 Yang et al., unpublished AY864057 Herbertetal.,unpublished AY968449 Zhangetal.,unpublished Plant sequence accession number Bacterial sequence accession number Table 1: Continued. CaNZ1 KC796595 KC796604 KC796542 KC796511 KC796581 This study CaNZ3 KC796544 KC796513 KC796583 This study CaNZ2 KC796543 KC796512 KC796582 This study CmicMx1 KC796596 KC796606 KC796547 KC796514 KC796587 This study CmicMx3 KC796549 KC796516 KC796589 This study CmicMx2 KC796548 KC796515 KC796588 This study Nodule labels /64 m 󸀠󸀠 18.07 󸀠 /2400 m 󸀠 41 ∘ 30 ∘ ,19 󸀠 ,+173 󸀠󸀠 30 ∘ 42.24 󸀠 23 ∘ 42 Mexico Morelos, 99 New Zealand Hapukuriver,NorthCanterbury,Southisland: − microphylla . Species Locality coordinates/altitude (asl) C. arborea C C. intermedia C. terminalis C. ruscifolia C. sarmentosa C. papuana Datisca glomerata Casuarina equisetifolia BioMed Research International 5

Table 2: Primers used for PCR amplification and DNA sequencing.

󸀠 󸀠 Gene primers Sequence (5 -3 ) Amplicons approximate size (bp) References glnA DB41 TTCTTCATCCACGACCCG 500 (Clawson et al., 2004 [4]) DB44 GGCTTCGGCATGAAGGT dnaA F7154 dnaAF GAGGARTTCACCAACGACTTCAT 700 Bautista et al. unpublished F7155 dnaAR CRGAAGTGCTGGCCGATCTT IGS nif D-K F9372 nifD1 5 GTCATGCTCGCCGTCGGNG 700 This study F9374 nifK1 5 GTTCTTCTCCCGGTAyTCCCA

F9373 nifD2 5 ACCGGCTACGAGTTCGCNCA 700 This study F9375 nifK2 5 TGCGAGCCGTGCACCAGNG 18S-ITS1-5.8S-ITS2-28S ITS1 TCCGTAGGTGAACCTGCGG 700 (White et al., 1990 [52]) ITS4 TCCTCCGCTTATTGATATGC

F9030-CJ-ITSF AGCCGGACCCGCGACGAGTTT 400 This study F9031-CJ-ITSR CGACGTTGCGTGACGACGCCCA matK F9249-matKF ACATTTAAATTATGTGTCAG 700 This study F9250-matkR TGCATATACGCACAAATC root nodules used in this study yielded PCR-amplifiable DNA microbial side, the New Zealand microsymbionts were at forbothbacterialandplantPCRtargetsequencesinallcases. the root (Group A); then three groups emerged, group However, in several instances it was easier to amplify Frankia B comprising the Pakistani, Mexican, and Mediterranean than Coriaria DNA, which may have been mostly due to the symbionts from France, group C comprising microsym- specificity of the primer sets used. Thus, in this study, new bionts from Morocco, and then group D comprising French primers were designed (Table 2). and Japanese microsymbionts as well as the Dg1 reference For the bacterial microsymbionts, the average uncorrec- sequence obtained initially from a Pakistani soil. On the ted p-distances (proportion of differences between sequen- host plant side, group 1 at the root comprises New Zealand ces) were computed for each region and were found to be and South American sequences, while group 2 comprises the relatively small for dnaA(𝑝 = 0.0378), intermediate for glnA Japanese, Mediterranean, and Pakistani sequences. (𝑝 = 0.0625), and high for IGS nif D-K region (𝑝 = 0.0833). On the other hand, no significant correlations were found 2 Blastanalysesoftheindividualgenespermittedassigning for Frankia symbionts (𝑟 = 0.772; Fgeneticdist = (geogdist × −06 −02 2 them all to Frankia cluster 2. Nearly 3000 nucleotides were 5.830E ) + 2.541E )norfortheCoriaria host plants (𝑟 −06 −03 obtained by concatenating sequences of the three DNA = 0.883; Fgeneticdist = (geogdist × 2.023E )+6.460E ) regions. (data not shown). Sequences variation for Coriaria species was small based on matKgene(𝑝 = 0.0205) compared to ITS1-ITS2 sequences (𝑝 = 0.0423). By concatenating matK and ITS1- ITS2 region, 4. Discussion a composite sequence of 1500 nt was used for phylogenetic Cospeciation has been postulated to have occurred in inference. some Frankia actinorhizal host plants, in particular in the All studied sequences were analyzed independently to test Casuarina-Frankia cluster 1b [18]butnotinAlnus-infective for incongruence between the data sets for each symbiotic and Elaeagnus-infective Frankia strains where many isolates partner. Similar topologies have been generally observed able to fulfill Koch’s postulates have been obtained. To between phylogenetic trees inferred from glnA, dnaA, and test if cospeciation was general or an exception, it was IGS nif D-K sequences for Frankia and from matKandITS decided to study uncultured Frankia microsymbionts and sequences for Coriaria regardless of the used phylogenetic representative Coriaria hosts, a lineage where no Frankia methods (not shown). isolate exists and where geographic discontinuities may have The topologies of the trees obtained for the two symbiotic limited dispersion. DNA sequences were obtained from root partners were not congruent (Figure 1). Moreover, global nodules collected from New Zealand (C. arborea), Pakistan distance-based ParaFit analysis recovered mostly random (C. nepalensis), Japan (C. japonica), Mexico (C. microphylla), associations between Frankia and Coriaria host plant species andFranceandMorocco(C. myrtifolia) and multiple molec- (𝑝 = 0.33) and rejected cospeciation hypothesis. On the ular markers were analyzed for phylogenetic inference. 6 BioMed Research International

F.CjJB CmM1a 69 F.CjJA CmM2a 100 Dg1 99 CmMs1 F.CmF1 CmF2 99 F.CmF2 CmNy5 86 68 F.CmMs2 76 2 Group Group D Group C. myrtifolia 92 F.CmMs4 C. myrtifolia F.CmMs1 CnP1 90 89 F.CmM1a C. nepalensis 75 80 F.CmM1c C. terminalis 54 F.CmM1b C. intermedia

Group C Group F.CmNy4 C. japonica F.CmMs3 96 CjJA 75 F.CmM2b 70 81 F.CmNy1 CmicMx1 90 91 F.CmNy5 C. microphylla 99 F.CnP1 C. papuana Group B Group 99 87 F.CnP2 CaNZ1 90 97 F.CmMx1 C. arborea F.CmMx2 51 100 C. ruscifolia 1 Group 73 F.CaNZ2 C. sarmentosa 83

Group A Group F.CaNZ1 100 C. lurida 70 Frankia Coriaria

Oceania Europe/N. Africa Asia America

Figure 1: Phylogenetic trees of the Frankia microsymbionts (left) and the Coriaria host plants (right). The Frankia tree was constructed using the glnA, dnaA, and the nif D-K intergenic spacer, while the Coriaria tree was done using the matK and the 18S rRNA-ITS1-5.8S rRNA-ITS2- 28S rRNA with ML method using strain CcI3 and Casuarina as outgroups respectively for Frankia and hot plant phylogenetic trees. The numbers at branches indicate bootstrap results above 50%. Lines are drawn between the microsymbionts and their hosts. The color code indicates the place of origin of the leave or of the set when homogenous. The groups numbers 1 and 2 on the right are according to Yokoyama et al. [19].

Paleontological data based on macrofossils and pollen South American C. ruscifolia and C. microphylla species was fossils have brought several authors [36–40]toconcludethat contrary to that of Yokoyama et al. [19] who found the the Coriariaceae had a Laurasian origin (North America Eurasian species at the base using rbcL(alargesubunitof and Eurasia). There have been a few dissenting opinions, in ribulose 1,5-bisphosphate carboxylase/oxygenase) and matK particular those of Croizat [41]andSchuster[42]whocon- (maturase K) genes. The present study suggests that the sidered that Coriaria originated in Gondwana and migrated Coriaria ancestor may have emerged between Asia and NZ to the Northern Hemisphere. However, such paleontological andthendispersedworldwideandthattheAsianlineage studies are not very convincing, as it is recognizably hard may have given rise relatively recently to the Mediterranean to ascribe fossils to a given family and even more so to a species,whiletheNZlineagegaverisetotheNorthAmerican given genus. Thus, several authors have been surprised by species (Figure 2). the results of molecular phylogeny positioning Coriariaceae Previous studies had concluded that Frankia cluster 2 had close to the Datiscaceae. Molecular approaches would thus alowgeneticdiversity[6, 7, 16] but these studies had been give support to a Gondwanan origin. focusedononlypartofthefulldiversityofthesymbiotic Yokoyama et al. [19]proposedthatCoriaria species had Coriaria-Frankia, essentially in North America and Mediter- emerged 59–63 million years ago, which is coherent with ranean.Inthisworkweaimedtoexpandthescopeofthe the date of 70 million years proposed by Bell et al. [25], study to the worldwide diversity and phylogeny of microsym- considerably older than that proposed (30 million years) by bionts of Coriaria species. Four microbial subgroups were thesameauthorsfortheCasuarinaceae. identified that did not fit to the geographic range of the host Topology and clustering of Coriaria phylogeny obtained plants, while two host plant subgroups were identified. The in the current study are similar to those obtained by position of subgroup A containing microsymbionts of New Yokoyama et al. [19],whilethepositionatthebaseofthe Zealand C. arborea at the base of Frankia cluster2isin host plant species from New Zealand, C. arborea, and the agreement with previous study [16]. In view of previously BioMed Research International 7

CmNy1-2-3-4-5 CmF1-2-3-4-5 Coriaria myrtifolia CmM1a-b-c 2 CmM a-b C. nepalensis C. japonica CmMs1-2-3-4-5 CnP1-2-3 CjJA-B-C-D-E C. terminalis C. microphylla C. intermedia CmicMx1-2-3

C. papuana Coriaria sp.

C. sarmentosa

C. ruscifolia Coriaria agustissima C. arborea CaNZ1-2-3 C. kingiana C. lurida C. plumosa C. pottsiana C. pteroides C. sarmentosa

Figure 2: Distribution of Coriaria species. Root nodules have been sampled from C. myrtifolia, C. arborea, C. nepalensis, C. japonica, and C. microphylla growing in Mediterranean areas (Morocco and France), New Zealand, Pakistan, Japan, and Mexico, respectively. Short arrows indicate sampling sites for this study while long arrows indicate possible routes of dispersal as discussed. reported data, members of cluster 2 Frankia studied here occurred or if there is survival of Frankia cluster 2 in the were found to have relatively higher sequences variation (p- rhizosphere of nonhosts as was recently demonstrated for distance = 0.0625) than those reported by Vanden Heuvel et Alnus glutinosa in Tunisia [45]. The numerous transitions al. [16](𝑝 = 0.00454) based on the same 460 nt of the glnA seen in the Frankia phylogenetic tree from one continent to gene. another would reinforce the idea. Molecular clock dating suggests that Frankia genus has Yokoyama et al. [19] concluded from their study of the emerged much earlier, 125 Myr bp before the appearance of Coriaria species phylogeny that the Eurasian species had angiosperm fossils in the Cretaceous period and the extant diverged earlier and are more diverse than other groups, but actinorhizal plants [4]. Normand et al. [5]usingthe4% that nevertheless the origin of the genus could have been in divergence in the 16S rRNA between cluster 2 and other North America, whence the South America and the Pacific Frankia lineages as equivalent to 50 MY/1% distance [17] species could have originated. Our study brings us to suggest concluded that the genus Frankia had emerged long before a third possibility, Oceania, which could also be the origin the extant dicotyledonous lineages. These authors proposed of this actinorhizal symbiosis, which can be concluded from Frankia cluster 2 as the proto-Frankia as nonsymbiotic phylogenetic inferences positioning both bacterial and host ancestor of 62–130 Myr bp [43]and100–200Myrbp[5]. Since plant partners as at the base to Frankia-Coriaria symbiosis. the distance in the 16S rRNA gene between cluster 1a (Frankia Another element that would support this hypothesis is the alni)andcluster1bislessthan1%,thedateofemergenceofthe large number of extant species there; according to Yokoyama Casuarina-infective lineage has been proposed to be less than et al. [19] New Zealand would be home to 8 of the 17 existing 50 million years [5]. Thus the Casuarina/Frankia 1b lineage is species. A similar argument has often been made to establish considerably younger than the Coriaria/Frankia lineage and Sub-Saharan Africa as the cradle of humankind [46]or wouldhavehadlesstimetomigrateoutofitscradleand Mexico for maize [47]. mingle with other hosts in its new territories and lose the Comparison of both the plant and the microbe phyloge- cospeciation signal. netic topologies did not show any evidence for cospeciation Symbiotic partnership often tends to become obligatory, of Frankia microsymbiontsand their Coriaria host species. as in the case of Casuarina host plants, where Frankia is only The results obtained in this study suggest that Frankia present in soils close to the host plant [44], which means that microsymbionts hosted currently by Coriaria species had the bacterium loses autonomy and becomes dependent on its probably dispersed globally as a proto-Frankia,afreeliving host. Speciation of the host could then lead to synchronous and nonsymbiotic ancestor. In parallel, the proto-Coriaria speciation of its microsymbiont unless dispersal through then diversified into the extant Coriaria species that appear to long-distance carriers such as winds or migratory birds have been retreating given their scattered distribution, a trend 8 BioMed Research International possibly reinforced recently due to man uprooting because nodule symbioses with Frankia 16S rRNA and glutamine syn- of the toxicity of the fruits for mammals [48, 49]. It can thetase gene sequences,” Molecular Phylogenetics and Evolution, thus be hypothesized that Coriaria appeared in the Pacific vol.31,no.1,pp.131–138,2004. Islands more than 70 million years ago and presumably was [5] P.Normand, S. Orso, B. Cournoyer et al., “Molecular phylogeny symbiotic from the start, before dispersing over all continents of the genus Frankia and related genera and emendation of as they drifted apart. The Coriaria species diversified in the family Frankiaceae,” International Journal of Systematic their different biotopes, as they saw the appearance of other Bacteriology,vol.46,no.1,pp.1–9,1996. plants hosting the same microsymbiont of Frankia cluster 2 [6] F. Ghodhbane-Gtari, I. Nouioui, M. Chair, A. Boudabous, and such as Datiscaceae,Rosaceae,Ceanothus,orevennonhost M. Gtari, “16S-23S rRNA intergenic spacer region variability in species such as Alnus glutinosa that was recently found to the genus Frankia,” Microbial Ecology,vol.60,no.3,pp.487– 495, 2010. host Frankia cluster 2 in its rhizosphere [45]. Members of these alternative host plant species cooccur sympatrically [7] I. Nouioui, F. Ghodhbane-Gtari, N. J. Beauchemin, L. S. Tisa, and M. Gtari, “Phylogeny of members of the Frankia genus with Coriaria such as Ceanothus and Purshia species in based on gyrB, nifH and glnII sequences,” Antonie van Leeuwen- Mexico and Datisca cannabina in Pakistan. These Frankia hoek,vol.100,no.4,pp.579–587,2011. cluster 2 host plant species have more extended geographic [8]P.NormandandD.R.Benson,“GenusIFrankia Brunchorst distribution and overlap in some instances Coriaria’s disjunct 1886, 174AL,” in Bergey’s Manual of Systematic Bacteriology, The area and as a result can compensate Frankia microsymbionts Actinobacteria,M.Goodfellow,P.Kampfer,¨ H.-J. Busse et al., remoteness, which would thus obscure the cospeciation Eds., vol. 5 of Bergey’s Manual Trust,pp.512–520,Springer,New signal. Cospeciation may also occur but subsequently is lost York, NY, USA, 2012. after bacterial mixing and fitness selection in the presence of [9] J. G. Torrey, “Cross-inoculation groups within Frankia and “indigenous” and “dispersal” symbionts. host-endosymbiont associations,” in The Biology of Frankia and Actinorhizal Plants,C.R.SchwintzerandJ.D.Tjepkema,Eds., pp. 83–106, Academic Press, San Diego, Calif, USA, 1990. Conflict of Interests [10] S. J. Kohls, J. Thimmapuram, C. A. Buschena, M. W. Paschke, and J. O. Dawson, “Nodulation patterns of actinorhizal plants The authors declare that there is no conflict of interests in the family Rosaceae,” Plant and Soil,vol.162,no.2,pp.229– regarding the publication of this paper. 239, 1994. [11] M. S. Mirza, K. Pawlowski, F. Y. Hafeez, A. H. Chaudhary, Acknowledgments andA.D.L.Akkermans,“Ultrastructureoftheendophyteand localization of nifH transcripts in root nodules of Coriaria This work is supported by CMCU (ComiteMixteTuniso-´ nepalensis Wall. by in situ hybridization,” New Phytologist,vol. Franc¸ais pour la Cooperation´ Inter-Universitaire No. 126,no.1,pp.131–136,1994. 10/G0903).TheauthorsaregratefultoDr.Mar´ıa Valdes´ [12]G.Nick,E.Paget,P.Simonet,A.Moiroud,andP.Normand, (Escuela Nacional de Ciencias Biologicas,´ Mexico,´ Mexico),´ “The nodular endophytes of Coriaria spp. form a distinct Dr. Sajjad Mirza (National Institute for Biotechnology lineage within the genus Frankia,” Molecular Ecology,vol.1,no. Genetic Engineering, Faisalabad, Pakistan), Dr. Warwick 3, pp. 175–181, 1992. Silvester (University of Waikato, Waikato, New Zealand), [13] M. Bosco, S. Jamann, C. Chapelon, and S. P.Normand, “Frankia Dr. Kawther Benbrahim and Dr. A. Ennabili (University of microsymbiont in Dryas drummondii nodules is closely related to the microsymbiont of Coriaria and genetically distinct from Fes, Fes, Morocco), Mr. Spick (Montpellier Botanical garden, other characterized Frankia strains,” in Nitrogen Fixation with France),Dr.J.C.Cleyet-Marel(MontpellierINRA,France), Non-Legumes,N.A.Hegazi,M.Fayez,andM.Monib,Eds.,pp. Mr. D. Moukouanga (IRD Montpellier, France), and Dr. 173–183, The American University in Cairo Press, 1994. Takashi Yamanaka (Forest and Forestry Products Research [14] D. R. Benson, D. W. Stephens, M. L. Clawson, and W. B. Institute, Ibaraki, Japan) for providing Coriaria nodules. Silvester, “Amplification of 16S rRNA genes from Frankia strains in root nodules of Ceanothus griseus, Coriaria arborea, Coriaria plumosa, Discaria toumatou,andPurshia tridentata,” Applied References and Environmental Microbiology,vol.62,no.8,pp.2904–2909, [1] M. P.Lechevalier, “Taxonomy of the genus Frankia (Actinomyc- 1996. etales),” International Journal of Systematic Bacteriology,vol.44, [15] D. R. Benson and M. L. Clawson, “Evolution of the actinorhizal no. 1, pp. 1–8, 1994. plant symbioses,” in Prokaryotic Nitrogen Fixation: A Model System for Analysis of Biological Process,E.W.Triplett,Ed.,pp. [2] D. R. Benson, B. D. Vanden Heuvel, and D. Potter, “Actinorhizal 207–224, Horizon Scientific Press, Wymondham, UK, 2000. symbioses: diversity and biogeography,” in Plant Microbiology, M. Gillings, Ed., pp. 97–127, BIOS Scientific Publishers, Oxford, [16] B. D. Vanden Heuvel, D. R. Benson, E. Bortiri, and D. Potter, UK, 2004. “Low genetic diversity among Frankia spp. strains nodulating sympatric populations of actinorhizal species of Rosaceae, [3]M.Gtari,L.S.Tisa,andP.Normand,“DiversityofFrankia Ceanothus (Rhamnaceae) and Datisca glomerata (Datiscaceae) strains, actinobacteria symbionts of actinorhizal plants,” in west of the Sierra Nevada (California),” Canadian Journal of Symbiotic Endophytes, Soil Biology,R.Aroca,Ed.,vol.37, Microbiology, vol. 50, no. 12, pp. 989–1000, 2004. Chapter 7, pp. 123–148, Springer, Berlin, Germany, 2013. [17] H. Ochman and A. C. Wilson, “Evolution in bacteria: evidence [4]M.L.Clawson,A.Bourret,andD.R.Benson,“Assessingthe for a universal substitution rate in cellular genomes,” Journal of phylogeny of Frankia-actinorhizal plant nitrogen-fixing root Molecular Evolution,vol.26,no.1-2,pp.74–86,1987. BioMed Research International 9

[18] P. Simonet, E. Navarro, C. Rouvier et al., “Co-evolution [37] J. A. Doyle, “Cretaceous angiosperm pollen of the Atlantic between Frankia populations and host plants in the family coastal plain and its evolutionnary significance,” Journal of the Casuarinaceae and consequent patterns of global dispersal,” Arnold Arboretum,vol.50,pp.1–35,1969. Environmental Microbiology,vol.1,no.6,pp.525–533,1999. [38] P. H. Raven and D. I. Axelrod, “Angiosperm biogeography and [19] J. Yokoyama, M. Suzuki, K. Iwatsuki, and M. Hasebe, “Molecu- past continental movements,” Annals of the Missouri Botanical lar phylogeny of Coriaria, with special emphasis on the disjunct Garden,vol.61,pp.539–673,1974. distribution,” Molecular Phylogenetics and Evolution,vol.14,no. [39] J. Muller, “Fossil pollen records of extant angiosperms,” The 1, pp. 11–19, 2000. Botanical Review,vol.47,no.1,pp.1–142,1981. [20] R. D. O. Good, “The geography of the genus Coriaria,” New [40] O. Eriksson and B. Bremer, “Pollination systems, dispersal Phytologist,vol.29,pp.170–198,1930. modes, life forms, and diversification rates in angiosperm [21] H. H. Allan, “Coriariaceae,” in Flora of New Zealand,L.B. families,” Evolution,vol.46,pp.258–266,1992. Moore, Ed., pp. 300–305, Government printer, Wellington, New [41] L. Croizat, Manual of Phytogeography: An Account of Plant Zealand, 1961. Dispersal Throughout the World, Junk, The Hague, 1952. [22] R. Melville, “Continental drift, mesozoic continents and the [42] R. M. Schuster, “Plate tectonics and its bearing on the geograph- migrations of the angiosperms,” Nature,vol.211,no.5045,pp. ical origin and dispersal of angiosperms,” in Origin and Early 116–120, 1966. Evolution of Angiosperms, C. B. Beck, Ed., pp. 48–138, Columbia University Press, New York, 1976. [23] L. E. Skog, “The genus Coriaria Coriariaceae in the Western [43] S.-C. Jeong, A. Liston, and D. D. Myrold, “Molecular phylogeny Hemisphere,” Rhodora,vol.74,pp.242–253,1972. of the genus Ceanothus (Rhamnaceae) using rbcLandndhF [24] R. Melville, “Vicarious plant distributions and paleogeography sequences,” Theoretical and Applied Genetics,vol.94,no.6-7,pp. of the Pacific region,”in Vicariance Biogeography,G.Nelsonand 852–857, 1997. E. D. Rosen, Eds., pp. 413–435, Columbia University Press, New [44] J. F. Zimpfer, C. A. Smyth, and J. O. Dawson, “The capacity of York, NY, USA, 1981. Jamaican mine spoils, agricultural and forest soils to nodulate [25]C.D.Bell,D.E.Soltis,andP.S.Soltis,“Theageanddiversi- Myrica cerifera, Leucaena leucocephala and Casuarina cunning- fication of the angiosperms re-revisited,” American Journal of hamiana,” Physiologia Plantarum,vol.99,no.4,pp.664–672, Botany,vol.97,no.8,pp.1296–1303,2010. 1997. [26] C. Rouvier, J. Schwenke, Y. Prin et al., “Biologie et diversite´ [45] I. Nouioui, I. Sbissi, F. Ghodhbane-Gtari, K. Benbrahim, P. gen´ etique´ des souches de Frankia associees´ aux Casuarinacees,”´ Normand, and M. Gtari, “First report on the occurrence of the Acta Botanica Gallica,vol.143,pp.567–580,1996. uncultivated cluster 2 Frankia microsymbionts in soil outside [27] J. D. Thompson, D. G. Higgins, and T. J. Gibson, “CLUSTAL the native actinorhizal host range area,” Journal of Biosciences, W: improving the sensitivity of progressive multiple sequence vol. 38, pp. 695–698, 2013. alignment through sequence weighting, position-specific gap [46] S. Ramachandran, O. Deshpande, C. C. Roseman, N. A. penalties and weight matrix choice,” Nucleic Acids Research,vol. Rosenberg, M. W. Feldman, and L. L. Cavalli-Sforza, “Support 22, no. 22, pp. 4673–4680, 1994. from the relationship of genetic and geographic in human [28] K. Tamura, D. Peterson, N. Peterson, G. Stecher, M. Nei, and populations for a serial founder effect originating in Africa,” S. Kumar, “MEGA5: molecular evolutionary genetics analysis Proceedings of the National Academy of Sciences of the United using maximum likelihood, evolutionary distance, and max- States of America,vol.102,no.44,pp.15942–15947,2005. imum parsimony methods,” Molecular Biology and Evolution, [47] B. Prasanna, “Diversity in global maize germplasm: character- vol. 28, no. 10, pp. 2731–2739, 2011. ization and utilization,” Journal of Biosciences,vol.37,pp.843– [29] M. Kimura, “Asimple method for estimating evolutionary rates 855, 2012. of base substitutions through comparative studies of nucleotide [48] L. De Haro, P.Pommier, L. Tichadou, M. Hayek-Lanthois, and J. sequences,” Journal of Molecular Evolution,vol.16,no.2,pp.111– Arditti, “Poisoning by Coriaria myrtifolia Linnaeus: a new case 120, 1980. report and review of the literature,” Toxicon,vol.46,no.6,pp. [30] N. Saitou and M. Nei, “The neighbor-joining method: a new 600–603, 2005. method for reconstructing phylogenetic trees,” Molecular Biol- [49] S. F. Belcher and T. R. Morton, “Tutu toxicity: three case ogy and Evolution,vol.4,no.4,pp.406–425,1987. reports of Coriaria arborea ingestion, review of literature and recommendations for management,” The New Zealand Medical [31] J. Felsenstein, “Confidence limits on phylogenies: an approach Journal,vol.126,pp.103–109,2013. using the bootstrap,” Evolution,vol.39,pp.783–791,1985. [50] T. Persson, D. R. Benson, P. Normand et al., “Genome [32]F.RonquistandJ.P.Huelsenbeck,“MrBayes3:bayesianphylo- sequence of “Candidatus Frankia datiscae” Dg1, the uncultured genetic inference under mixed models,” Bioinformatics,vol.19, microsymbiont from nitrogen-fixing root nodules of the dicot no. 12, pp. 1572–1574, 2003. Datisca glomerata,” Journal of Bacteriology,vol.193,no.24,pp. [33] P. Legendre, Y. Desdevises, and E. Bazin, “A statistical test for 7017–7018, 2011. host-parasite coevolution,” Systematic Biology,vol.51,no.2,pp. [51]P.Normand,P.Lapierre,L.S.Tisaetal.,“Genomecharacter- 217–234, 2002. istics of facultatively symbiotic Frankia sp. strains reflect host [34] J. P. Meier-Kolthoff, A. F. Auch, D. H. Huson, and M. Goker,¨ range and host plant biogeography,” Genome Research,vol.17, “CopyCat: cophylogenetic analysis tool,” Bioinformatics,vol.23, no.1,pp.7–15,2007. no. 7, pp. 898–900, 2007. [52] T. J. White, T. Bruns, S. Lee, and J. Taylor, “Amplification [35] R. Ihaka and R. Gentleman, “R: a language for data analysis and and direct sequencing of fungal ribosomal RNA genes for graphics,” JournalofComputationalandGraphicalStatistics,vol. phylogenetics,” in PCR Protocols: A Guide to Methods and 5, no. 3, pp. 299–314, 1996. Applications, M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. [36] A. N. Gladkova, “Fragments of the history of the Myricaceae J. White, Eds., pp. 315–322, Academic Press, San Diego, Calif, family,” Pollen and Spore,vol.4,p.345,1962. USA, 1990.